Submitted by Sana Rahim

Synthesis and Characterization of Homo and Amphiphilic

Block Copolymers of Poly(2-vinylpyridine) Stabilized

Metallic Nanoparticles

Submitted by

Sana Rahim

Dissertation for the Partial Fulfilment of the Degree of

Doctor of Philosophy

H. E. J. Research Institute of Chemistry,

International Center for Chemical and Biological Sciences,

University of Karachi, Karachi-75270, Pakistan.

2018

ii

CERTIFICATE

To Whom It May Concern

It is certified that the thesis entitled, ―Synthesis and Characterization of Homo and

Amphiphilic Block Copolymers of Poly(2-vinylpyridine) Stabilized Metallic Nanoparticles”,

submitted to the Board of Advance Studies and Research, University of Karachi, by Ms. Sana

Rahim, satisfies the requirements for the Ph.D. degree in Chemistry.

Dr. Muhammad Imran Malik

Supervisor



University of Karachi, Karachi, Pakistan

Dr. Muhammad Raza Shah (T.I.)

Co-supervisor



University of Karachi, Karachi, Pakistan

iii

DEDICATION

Dedicated to My Beloved Parents,

Mr. Rahim Gul

&

Mrs. Madiha

iv

ABSTRACT

This Ph. D. dissertation deals with the synthesis of metallic nanoparticles with P2VP homo-

and block copolymers (BCP) as stabilizing agent, their characterization, and applications. As

a summary of the research conducted during the course of Ph. D., a series of homopolymers

of poly(2-vinylpyridine) and amphiphilic block copolymers of poly(2-vinylpyridine),

including poly(2-vinylpyridine)-block-poly(methyl methacrylate) and polystyrene-block-

poly(2-vinylpyridine) were used to stabilize metallic nanoparticles (gold and silver).

Polymers containing pyridine moiety have been utilized as a stabilizing agent for the

metallic nanoparticles. Among them poly(2-vinylpyridine) (P2VP) is the excellent

candidates because nitrogen atoms of the pyridine moiety have a strong affinity for the

metal ions and metallic nanoparticles that restrains the aggregation of the metal

nanoparticles through steric stabilization. Furthermore, P2VP prompts the reaction at

ambient temperature and reduction of the particle size with the increase in its molar mass.

AuNPs stabilized by P2VP ligands were designed to offer atomic level control and an

efficient scale-up production through control of the molar mass of P2VP. Molar mass of

the P2VP has enormous effect on the stabilization, size and size distribution of AuNPs.

The reducing activity of P2VP increased with the increase in its molar mass. The P2VP

stabilized AuNPs are evaluated for their stability and applications using UV-visible

spectrophotometry, FTIR, DLS and AFM. Moreover, the drug encapsulation efficiency of

P2VP-stablized AuNPs increased with the molar mass of P2VP.

P(S-VP)-AgNPs were used as nanosensor for the rapid quantitative assay of pesticide,

cartap. P(S-VP)-AgNPs and its interaction with cartap was studied using UV-visible

spectroscopy, FTIR, zetasizer and AFM. The synthesized nanosensor is selective towards

cartap in the presence of other interfering pesticides in real samples. The LoD of the

nanosensor for cartap is far below already reported sensors for cartap.

Furthermore, P(2VP-MMA)-AuNPs modified GCE electrode was used as a novel

electrochemical sensor for nicotine. It was found that sensitivity of bare GCE is

v

significantly enhanced by coating with P(2VP-MMA)-AuNPs. The P(2VP-MMA)-

AuNPs modified GCE is more sensitive towards nicotine and gave more intense

electrochemical response with reference to bare GCE.

In addition, the morphology of P(2VP-MMA) copolymer thin films was studied using

AFM. It was observed that both P2VP and PMMA block lengths, total molar mass of

block copolymer, solvent used for casting, and substrate play an important role in the

morphology of block copolymer thin film. Gold nanoparticles incorporated with the

polymer are completely shielded by P2VP chains and influenced the morphology of

block copolymer organization by enlarging the polymer domain. Furthermore, surface

roughness and thickness increased with the increase in molar mass of the block

copolymers.

vi

KHULASA

vii

viii

ACKNOWLEDGEMENTS

I am gratified to Almighty Allah who endowments me the power and courage to fulfil all

my tasks. Primarily, I am thankful to the H.E.J. Research Institute of Chemistry

(I.C.C.B.S), University of Karachi, for providing all the research facilities, infrastructure

and also the financial support for the successful completion of the current dissertation. I

pay to thanks its great pillars Prof. Dr. Atta ur Rahman (FRS, N.I., H.I., S.I., T.I.), and

Prof. Muhammad Iqbal Choudhary (H.I., S.I., T.I.), who are keen to improve its

standards, and their breathtaking leadership to make it one of the finest academic

establishments in the developing world.

It is indeed the greatest pleasure to extend my gratitude to my supervisor, Dr. Muhammad

Imran Malik and co-supervisor Prof. Dr. Muhammad Raza Shah (T.I.), for their

cooperation, reinforcement and advices. They become a source of inspiration and role

model for me to accomplish this task and to achieve my goals.

I would like to extend my thanks to all the collaborating groups; Dr. Muhammad Iqbal

Bhanger and Dr. Asma Rauf for their precious attention and providing a facility of cyclic

voltammetry and other lab staff.

All my research became possible due to the friendly environment and positive attitude of

my lab fellows, Muhammad Khurram Tufail, Rubina Abdul Karim, Adnan Murad,

Tehsin Ahmed, Sidra Safdar Durrani, Ayaz Anwar, Kiramat, Farid Ahmed, Dania

Ahmed, Sadia, Faiza, Zara Aslam, Shama Noureen, Imkaan and Imdad. I also very

thanks to my other friends Rabia Aslam, Saira Yasmeen, Ruqaiya Khalil for support,

kindness and most memorable moments. I am thankful to Mr. Hussain our lab assistant

for his help.

I have no words to express my appreciations for my beloved parents for their great

contribution in my life. I heartedly grateful for their cooperation, support and prayers. I

am also acknowledge my all siblings by my heart for their financial and moral support.

Sana Rahim,

Karachi, 2018

ix

CURRICULUM VITAE

I was born in Gujranwala (Punjab) Pakistan on 6th of March

1985, belonging to a middle class family. I started my formal

education career in Peshawar. My first year of education was too

much hard because I joined the school from class 2, without

going from the preprimary section and totally unfamiliar how to

write, read and learn the things in a class like other children did.

My teachers scolded and beated me every time on my mistakes,

therefore, I felt the embarrassment in front of other students and was afraid from the

name of teacher. I was little but decided to work hard and became like other children. I

made efforts and unimaginably after four to five months I was able to write, read and

learn all the stuff taught in the class. I think, it was the moment when I learnt how to

become a successful person in life.

After two years, my father was posted to Karachi, so I continued my further education

here in Karachi and passed the Matric examination from nearby school named, Shaheen

High School in 2000. I joined Khursheed Government Girls College for F. Sc. (Pre

medical) in 2001 and did B.Sc. from B.A.M.M P.E.C.H.S Government College For

Women in 2003. At that point, I discontinued my studies because of some family issues

but after 2 years, I got a degree of B.Ed. from Jamia Millia College Malir in 2008 and

then passed the M.Sc. (Analytical Chemistry) from University of Karachi, in 2010.

During M.Sc. in summer vacations, I had an opportunity of internship in Associated

Industries Limited, Nowshera, Pakistan and also after M.Sc. did internship in

multinational company Clariant Pakistan Limited, currently named as Archroma Pakistan

Limited.

Where I realized the importance of Ph.D. but unfortunately for two years I did not get the

chance to enroll.

In July 2013, I joined the polymer chemistry department of H.E.J. Research Institute of

Chemistry, International Center for Chemical and Biological Sciences, University of

x

Karachi, for Ph.D. studies under the supervision of Dr. Muhammad Imran Maik and co-

supervision of Dr. Muhammad Raza Shah. My research mainly devoted to the application

of polymer based metallic nanoparticles as a nanosensor and morphological studies of

polymer thin films using AFM. During my Ph.D. research, I have attended many

conferences and workshops and presented scientific findings as posters. I will always

remember my stay at this institute. It has been an extraordinary life so far. My hobbies

are reading books (historical books and scientific literatures) making drawing, watching

movies and listening music.

xi

LIST OF ABBREVIATIONS

Ae Auxiliary Electrode

AFM Atomic Force Microscopy

AgNPs Silver Nanoparticles

Amax Absorption Maxima

AuNPs Gold Nanoparticles

BCP Block Copolymer

CE Capillary Electrophoresis

CNTs Carbon Nanotubes

CV Cyclic Voltammetry

CV Cyclic Voltammetry

DLS Dynamic Light Scattering

DNA Deoxyribonucleic acid

DP Degree of Polymerization

DSC Differential Scanning Calorimeter

EMR Electromagnetic Radiations

FTIR Fourier Transform Infrared

FTIR Fourier Transformed Infrared

GC Gas Chromatography

GCE Glassy Carbon Electrode

GC-MS Gas Chromatography-Mass Spectrometry

HOPG Highly Oriented Pyrolytic Graphite

HPLC High Performance Liquid Chromatography

HPLC High Performance Liquid Chromatography

IRAC MoA Insecticide Resistance Action Committee Mode of Action

LC-MS Liquid Chromatography-Mass Spectrometry

LMW Low Molecular Weight

LOD Limit of Detection

Mn Number Average Molecular Weight

MNPs Metallic Nanoparticles

xii

Mp Molecular Weight at Peak Height

MRI Magnetic Resonance Imaging

Mw Molecular Weight

MWNT Multi-Walled Nanotube

NPs Nanoparticles

P2VP Poly(2-vinylpyridine)

P2VP-b-PMMA Poly(2-vinylpyridine)-block-poly(methylmethacrylate)

PDI Polydispersity Index

PMMA Poly (methyl methacrylate)

PRB Plasmon Resonance Band

PS Polystyrene

PS-b-P2VP Polystyrene-block-poly(2-vinylpyridine)

Re Reference Electrode

Rg Radius of gyration

RMS Root Mean Square

rpm Revolutions Per Minute

SE Supporting Electrolytes

SEM Scanning Electron Microscopy

Si Silicon

SLS Static Light Scattering

SPB Surface Plasmon Band

SPM Scanning Probe Microscopy

SPR Surface Plasmon Resonance

SWNT Single-Walled Nanotube

TEM Transmission Electron Microscopy

Tg Glass Transition Temperature

UV-vis Ultraviolet visible

We Working Electrode

Wep Working Electrode Potential

Zp Zeta potential

xiii

TABLE OF CONTENTS

CERTIFICATE ................................................................................................................... II

DEDICATION .................................................................................................................... III

ABSTRACT ......................................................................................................................... IV

KHULASA ........................................................................................................................... VI

ACKNOWLEDGEMENTS ........................................................................................... VIII

CURRICULUM VITAE .................................................................................................... IX

LIST OF ABBREVIATIONS ........................................................................................... XI

TABLE OF CONTENTS ................................................................................................ XIII

LIST OF FIGURES ...................................................................................................... XVIII

LIST OF TABLES ........................................................................................................ XXIII

LIST OF SCHEMES .................................................................................................... XXIV

CHAPTER 1 .......................................................................................................................... 1

GENERAL INTRODUCTION & LITERATURE REVIEW ........................................ 1

1. POLYMER .................................................................................................................... 2

1.1 PROPERTIES OF POLYMERS ..................................................................................2

1.1.1 Monomers and Repeating Units .....................................................................3

1.1.1.1 Block Copolymer ....................................................................................4

1.1.1.2 Amphiphilic Block Copolymer ...............................................................5

1.1.2 Microstructure ................................................................................................6

1.1.2.1 Polymer architecture ...............................................................................6

1.1.2.2 Chain length ............................................................................................6

1.1.3 Polymer Morphology ......................................................................................7

1.1.3.1 Crystallinity .............................................................................................7

1.1.3.2 Radius of gyration ...................................................................................7

1.1.4 Phase Behavior ...............................................................................................8

xiv

1.1.4.1 Melting point ...........................................................................................8

1.1.4.2 Glass transition temperature ....................................................................8

1.1.4.3 Mixing behavior ......................................................................................8

1.1.5 Chemical Properties ........................................................................................8

1.2 POLY(2-VINYL PYRIDINE) ....................................................................................9

1.3 POLYMER-METAL NANOMATERIALS .................................................................10

1.4 NANOPARTICLES ...............................................................................................12

1.4.1 Characteristics and Applications of Nanoparticles .......................................12

1.4.2 Quantum Confinement Effects .....................................................................13

1.4.3 Surface Plasmon Resonance (SPR) ..............................................................14

1.5 CLASSIFICATION OF NANOPARTICLES ...............................................................15

1.5.1 Zero dimensional nanoparticles ....................................................................15

1.5.2 One dimension nanoparticles ........................................................................16

1.5.3 Two dimension nanoparticles .......................................................................16

1.5.4 Three dimension nanoparticles .....................................................................16

1.6 METALLIC NANOPARTICLES ..............................................................................16

1.6.1 Gold Nanoparticles (AuNPs) ........................................................................18

1.6.2 Silver Nanoparticles (AgNPs) ......................................................................19

1.6.3 Other Nanoparticles ......................................................................................19

1.7 APPLICATIONS OF METALLIC NANOPARTICLES IN VARIOUS FIELDS .................19

1.7.1 Biomedicines ................................................................................................20

1.7.1.1 Drug delivery.........................................................................................20

1.7.2 Energy ...........................................................................................................20

1.7.3 Environment .................................................................................................20

1.7.3.1 Catalysis ................................................................................................20

1.7.3.2 Chemosensing .......................................................................................20

1.8 SYNTHETIC APPROACHES OF NANOPARTICLES..................................................21

1.8.1 Top-down approach ......................................................................................22

1.8.2 Bottom-up approach .....................................................................................22

1.9 LIMITATIONS IN NANOSCALE APPROACHES ......................................................23

1.10 CHARACTERIZATION OF NANOPARTICLES .........................................................23

xv

1.10.1 Particle Size and surface morphology ..........................................................24

1.10.1.1 Light scattering methods .......................................................................24

1.10.1.1.1 Dynamic Light Scattering (DLS)………………………………….25

1.10.1.1.2 Static Light Scattering (SLS)……………………………………...25

1.10.1.2 Scanning Electron Microscopy (SEM) .................................................25

1.10.1.3 Transmission Electron Microscopy (TEM) ..........................................26

1.10.1.4 Atomic Force Microscopy (AFM) ........................................................26

1.10.1.4.1 Contact Mode……………………………………………………..28

1.10.1.4.2 Tapping Mode…………………………………………………….28

1.10.1.4.3 Non-contact Mode………………………………………………...29

1.10.2 Surface Charge .............................................................................................30

1.10.3 Drug Loading/Releasing ...............................................................................30

1.10.3.1 High-Performance Liquid Chromatography (HPLC) ...........................30

1.10.3.2 UV-Visible Spectroscopy ......................................................................31

1.10.3.3 Fluorescence spectroscopy ....................................................................32

1.10.3.4 Fourier Transforms Infrared Spectroscopy (FTIR) ...............................34

1.10.3.5 Voltammetry..........................................................................................35

CHAPTER 2 ........................................................................................................................ 36

EVALUATION OF MORPHOLOGY, AGGREGATION PATTERN AND SIZE

DEPENDENT DRUG LOADING EFFICIENCY OF GOLD NANOPARTICLES

STABILIZED WITH POLY (2-VINYL PYRIDINE) ................................................... 36

ABSTRACT ......................................................................................................................... 37

2 INTRODUCTION ...................................................................................................... 37

2.1 EXPERIMENTAL .................................................................................................. 40

2.1.1 Materials and Instrumentation ....................................................................... 40

2.1.2 Preparation of P2VP Coated Gold Nanoparticles.......................................... 41

2.2 RESULTS AND DISCUSSION ................................................................................. 42

2.3 CONCLUSION ...................................................................................................... 55

CHAPTER 3 ........................................................................................................................ 56

xvi

POLYSTYRENE-BLOCK-POLY(2-VINYLPYRIDINE)-CONJUGATED SILVER

NANOPARTICLES AS COLORIMETRIC SENSOR FOR QUANTITATIVE

DETERMINATION OF CARTAP IN AQUEOUS MEDIA AND BLOOD PLASMA56

ABSTRACT ......................................................................................................................... 57

3 INTRODUCTION ...................................................................................................... 57

3.1 EXPERIMENTAL .................................................................................................. 60

3.1.1 Materials and Instrumentation ....................................................................... 60

3.1.2 Preparation of P2VP Coated Gold Nanoparticles.......................................... 61

3.1.3 Spiking in Tap Water and Surface Runoff Water .......................................... 61

3.1.4 Spiking in Human Blood Plasma................................................................... 61


3.2.1 Synthesis and characterization of P(S-VP)-AgNPs ....................................... 62

3.2.2 P(S-VP)-AgNPs and cartap response ............................................................ 65

3.2.3 Spectroscopic recognition of cartap .............................................................. 70

3.3 CONCLUSION ...................................................................................................... 79

CHAPTER 4 ........................................................................................................................ 80

ENHANCEMENT IN THE ELECTROCHEMICAL RESPONSE OF GLASSY

CARBON ELECTRODE MODIFIED BY POLY(2-VINLYPYRIDINE)-B-

POLY(METHYL METHACRYLATE) CONJUGATED GOLD NANOPARTICLES

FOR NICOTINE................................................................................................................. 80

ABSTRACT ......................................................................................................................... 81

4 INTRODUCTION ...................................................................................................... 81

4.1 EXPERIMENTAL SECTION ................................................................................... 84

4.1.1 Materials ........................................................................................................ 84

4.1.2 Instrumentation .............................................................................................. 84

4.1.3 Methods ......................................................................................................... 86

4.1.3.1 Preparation of P(2VP-MMA)-AuNPs .................................................... 86

4.1.3.2 Electrochemical studies .......................................................................... 87


xvii

4.2.1 Characterization of P(2VP-MMA)-AuNPs ................................................... 87

4.2.2 Cyclic Voltammetric detection of nicotine using P(2VP3-MMA97)-AuNPs-

GCE as a Sensor ....................................................................................................... 96

4.3 CONCLUSION .................................................................................................... 104

CHAPTER 5 ...................................................................................................................... 105

SELECTIVITY OF THIN FILMS OF POLY(2-VINYLPYRIDINE-BLOCK-

METHYL METHACRYLATE) COPOLYMERS: AN AFM STUDY .................... 105

ABSTRACT ....................................................................................................................... 106

5 INTRODUCTION .................................................................................................... 106

5.1 EXPERIMENTAL ................................................................................................ 108

5.1.1 Materials and Instrumentation ..................................................................... 108

5.1.2 Atomic Force Microscopy ........................................................................... 109

5.1.3 Sample Preparation ...................................................................................... 109

5.2 RESULTS AND DISCUSSION ............................................................................... 110

5.2.1 Characterization of Surface Morphology .................................................... 112

5.2.2 Effect of Casting Solvent ............................................................................. 117

5.2.3 Effect of Substrate ....................................................................................... 119

5.2.4 Thermal Annealing and Surface Morphology ............................................. 121

5.3 CONCLUSION .................................................................................................... 123

CHAPTER 6 ...................................................................................................................... 124

CONCLUSION ................................................................................................................. 124

REFERENCES ................................................................................................................. 128

LIST OF PUBLICATIONS ............................................................................................. 155

xviii

LIST OF FIGURES

Figure 1-1. Various kind of copolymers ..............................................................................3

Figure 1-2. Block copolymers having di-, tri- and multi-blocks .........................................4

Figure 1-3. Various schematic representations of shapes of block copolymers ..................5

Figure 1-4: Various shapes of different nanoparticles .......................................................12

Figure 1-5: Various kind of nanoparticles .........................................................................16

Figure 1-6: Synthetic approaches of nanoparticles ............................................................22

Figure 1-7: Process of light scattering in solution .............................................................24

Figure 1-8: Schematic representation of basic principle of AFM ......................................27

Figure 1-9: Contact mode of AFM ....................................................................................28

Figure 1-10: Tapping mode of AFM .................................................................................29

Figure 1-11: Non-contact mode of AFM ...........................................................................29

Figure 1-12: Schematic representation of basic instrumentation of UV-visible

spectrophotometer ..............................................................................................................31

Figure 1-13. Schematic representation of basic principle of fluorescence spectroscopy .34

Figure 2-1: Effect of molar mass of P2VP on size and stability of P2VP-stabilized

AuNPs; A) Colour of solution and size; B) UV-vis spectra ..............................................43

Figure 2-2: FTIR spectra of unstabilized AuNPs (> 10,000 nm), P2VP (5000 g/mol) and

P2VP-stabilized AuNPs .....................................................................................................44

Figure 2-3: AFM images of P2VP-stabilized AuNPs, showing the average particle sizes;

A) AuNPs/P2VP1K; 125 nm, B) AuNPs/P2VP2K; 96 nm, C) AuNPs/P2VP5K; 43 nm, D)

AuNPs/P2VP10K; 32 nm, E) AuNPs/P2VP20K; 28 nm. The scale bar represents 0.25 µm

on all images ......................................................................................................................45

Figure 2-4: Physical characterization of P2VP stabilized AuNPs by DLS. (A) Dynamic

light scattering results of P2VP2K illustrating the experimental conditions i.e., the mean

autocorrelation function, monodispersity and radius plot (I to III), respectively. (B)

xix

Comparative corresponding radius distribution of P2VP-stabilized AuNPs, effect of

molar mass on the size distribution. All experiments were performed with an auto–piloted

run of 50 measurements (20 s for single measurement) with a wait time of 1 s at 25 °C. 46

Figure 2-5: Stability of the P2VP-stabilized AuNPs as a function of residence time as

indicated by UV-vis spectroscopy .....................................................................................47

Figure 2-6: Effect of the concentration of P2VP5K on the stability, size and distribution of

AuNPs; A) Visual difference in colour, B) UV-Vis spectroscopy, C) Dynamic light

scattering ............................................................................................................................49

Figure 2-7: The effect of temperature on the stability of P2VP2K-stabilized AuNPs as

shown by UV-vis spectroscopy .........................................................................................50

Figure 2-8: Effect of pH on P2VP stabilized gold nanoparticles as shown by UV-vis

spectroscopy .......................................................................................................................51

Figure 2-9: Effect of various salt concentrations on P2VP coated gold nanoparticles as

shown by UV-vis spectroscopy, A) P2VP10K-Au NPs; B) P2VP2K-Au NPs .....................53

Figure 2-10: A) Calibration curves for quantification of Naringin in concentration range

of 0.00391-0.0625 mg/mL; B) % drug-loading efficiency of P2VP-stabilized AuNPs ....55

Figure 3-1. UV-visible spectrum of P(S-VP)-conjugated AgNPs .....................................63

Figure 3-2. UV-visible spectrum of P(S-VP)-conjugated AgNPs after incubation of P(S-

VP)-conjugated AgNPs at 64 °C for 10 minutes (B.P. of methanol) ................................64

Figure 3-3. Electrolyte effect on P(S-VP)-conjugated AgNPs with various salt

concentration (0.01mM-5M)..............................................................................................65

Figure 3-4. Schematic representation of cartap recognition of P(S-VP)-AgNPs through

electrostatic interactions.....................................................................................................66

Figure 3-5. The size distribution by intensity A) of P(S-VP)-AgNPs avg size: 104.2±0.68

nm, PDI: 0.22; B) P(S-VP)-AgNPs/ cartap. avg. size: 89.68±0.57 nm, PDI: 0.08 ..........66

Figure 3-6. Atomic force micrographs (AFMs) A) P(S-VP)-AgNPs (80-120 nm); B) P(S-

VP)-AgNPs/cartap (60-90 nm) ..........................................................................................67

xx

Figure 3-7. Zeta potential distribution A) P(S-VP)-AgNPs; B) P(S-VP)-AgNPs/Cartap .68

Figure 3-8. FTIR spectra of P(S-VP), Cartap, P(S-VP)-AgNPs, and cartap treated P(S-

VP)-AgNPs ........................................................................................................................69

Figure 3-9. UV-visible spectra of P(S-VP)-AgNPs complexed with various pesticides ...72

Figure 3-10: Effect of pH on accumulation of P(S-VP)-conjugated AgNPs with Cartap .73

Figure 3-11. A) UV-visible spactra by using various concentrations of cartap with P(S-

VP)-AgNPs; B) Calibration curve for amount of cartap at 410 nm ..................................74

Figure 3-12: Job‘s plot for binding ratio. ..........................................................................75

Figure 3-13. Effect of interfering pesticides on cartap detection by P(S-VP)-AgNPs, 1:

deltamethrin, 2: Alpha-cypermethrin, 3: carbofuran, 4: chlorfenapyr, 5: Lambda-

cyhlalothrin, 6: diuron, 7: imidacloprid, 8: lufenron, 9: clodinafop propa .......................76

Figure 3-14. Effect of cartap on absorbance intensity of P(S-VP)-AgNPs A) tap water; B)

surface runoff water; C) human blood plasma ...................................................................79

Figure 4-1. Schematic illustration of the reduction process of Au (III) particles in the

presence of a stabilizing block copolymer P(2VP-MMA) using NaBH4 as reducing agent. .. 86

Figure 4-2: UV-visible spectra of P(2VP-MMA)-AuNPs stabilized by different block

copolymers varying in total molar mass and chemical composition .................................88

Figure 4-3: Comparative FTIR spectra of P(2VP-MMA)-AuNPs, P(2VP-MMA) and

AuNPs ................................................................................................................................89

Figure 4-4: AFM images of P(2VP-MMA)-AuNPs ..........................................................90

Figure 4-5: Size distribution by intensity of P(2VP3-MMA97)-AuNPs, P(2VP15-MMA85)-

AuNPs, and P(2VP10-MMA90)-AuNPs. .............................................................................91

Figure 4-6: Zeta potential distribution P(2VP3-MMA97)-AuNPs, P(2VP15-MMA85)-

AuNPs, and P(2VP10-MMA90)-AuNPs ..............................................................................92

Figure 4-7: Time stability of P(2VP3-MMA97)-AuNPs (A) UV visible spectroscopy (B)

AFM. All the images are of 2x2µm ...................................................................................93

Figure 4-8: Temperature effect on P(2VP3-MMA97)-AuNPs ............................................94

xxi

Figure 4-9: Electrolyte effect on the stability of P(2VP3-MMA97)-AuNPs .......................95

Figure 4-10: pH effect on P(2VP3-MMA97)-AuNPs .........................................................96

Figure 4-11: Voltammetric response of nicotine on (a) bare GCE; (b) P(2VP3-MMA97)-

AuNPs, (c) P(2VP15-MMA85)-AuNPs, and (d P(2VP10-MMA90)-AuNPs. fabricated GCE.97

Figure 4-12: Cyclic voltammograms in the absence of nicotine on bare GCE while using

(a) acetonitrile (b) water as a solvent. ................................................................................98

Figure 4-13: A comparative view of cyclic voltammograms (a) absence (0 mM) and (b)

presence (0.05 mM) of nicotine on P(2VP3-MMA97)-AuNPs-GCE in acetonitrile. .........98

Figure 4-14: An overlay of (a) absence, (b) and (c) presence of nicotine on P(2VP3-

MMA97)-GCE in acetonitrile at scan rate of 0.1V.s-1

. .......................................................99

Figure 4-15: Cyclic voltammograms of nicotine with various concentrations ranging from

0.05 mM to 0.4 mM on bare GCE in (A) acetonitrile; (B) distilled deionized water. .....101

Figure 4-16. Effect of various concentrations (from 0.05 mM – 0.4 mM) of nicotine on

P(2VP3-MMA97)-AuNPs-GCE in acetonitrile. ................................................................101

Figure 4-17: Comparison of cyclic voltammograms of 0.4 mM nicotine on (a) bare GCE

and (b) P(2VP3-MMA97)-AuNPs-GCE and (c) P(2VP3-MMA97)-GCE-sensor in

acetonitrile........................................................................................................................102


and (b) P(2VP3-MMA97)-AuNPs-GCE in acetonitrile. ...................................................103

Figure 4-19: Plot of oxidative peak current as a function of concentration of nicotine (0.1

mM - 0.5 mM)..................................................................................................................103

Figure 5-1: Structure of poly(2-vinylpyridine-b-methylmethacrylate) ............................110

Figure 5-2: Schematic representation of micellization and self-organization of AuNPs in

P2VP domain ...................................................................................................................111

Figure 5-3: AFM topographical images of P(2VP-MMA) with schematic overview of the

fabrication of nanoporous layers by P(2VP-MMA) (A) P(2VP-MMA) copolymers with

different compositions (sample area, 10x10 µm) (B) P(2VP-MMA) copolymers with

xxii

different molecular weights (sample area, 10x10 µm). The scale bar on each image show

2.5 µm. .............................................................................................................................113

Figure 5-4: Morphology of P(2VP-MMA)-AuNPs .........................................................114

Figure 5-5: Amplitude roughness profile of (A) P(2VP-MMA), and (B) P(2VP-MMA)-

AuNPs at horizontal scale of 10x10 µm. .........................................................................115

Figure 5-6: AFM 3D phase images of P(2VP-MMA) and P(2VP-MMA)-AuNPs

showing the thickness of the film on the Si wafer. From top to the bottom: P(VP3-

MMA97), P(VP15-MMA85), P(VP10-MMA90) (left); P(VP3-MMA97)/AuNPs, P(VP15-

MMA85)/AuNPs and P(VP10-MMA90)/AuNPs, (right) ...................................................116

Figure 5-7: Comparison of P(2VP-MMA) and P(2VP-MMA)-AuNPs RMS values

obtained through AFM studies.........................................................................................117

Figure 5-8: Solvent effect on the surface morphology of P(2VP15-MMA85) on Si wafer119

Figure 5-9: AFM 3D phase images of P(2VP15-MMA85) block copolymers showing the

polymer-surface and polymer-air interaction of the film cast from chloroform on the

various substartes. ............................................................................................................120

Figure 5-10: Height profile of P(VP15-MMA85) on various substrates (A) HOPG (B) Si

wafer (C) Mica .................................................................................................................121

Figure 5-11: Effect of thermal annealing on the morphology of P(2VP15-MMA85)

copolymer film on Si wafer for 30 min. Scale bar on each image is 1µm ......................122

Figure 5-12: Thermal annealed films of P(2VP-MMA)-AuNPs on Si wafer for 30 min 123

xxiii

LIST OF TABLES

Table 1.1: Different characteristics and applications of nanoparticles ……………….…13

Table 1.2: Different characterization techniques for various parameters related to

nanoparticles……………………………………………………………………………..23

Table 2 1: Molecular weight and polydispersity index of P2VP homopolymers, as

provided by manufacturer …….………………………………………………….……...41

Table 3.1: Comparison of reported cartap detection methods with current study ……. 77

Table 4.1. Molecular weight and polydispersity index of P(2VP-MMA), as provided by

manufacturer………………………………………………………………………. 85

Table 5.1: Molecular weight and polydispersity index of P2VP-b-PMMA as provided by

manufacturer.…………………..……………………………….………………………109

xxiv

LIST OF SCHEMES

Scheme 3 1: Resonating structure of Cartap …………………………………………… 70

Scheme 3 2: Structure of cartap and other interfering pesticides ……………………… 71

1

Chapter 1

General Introduction & Literature Review

2

1. Polymer

Polymers are one of the essential parts of modern life. The list of the products and

accessories made of polymers for daily life includes clothes, dishes, bottles, cars,

computers, pens, lights etc. The applications of polymers are endless to the high

technology such as spaceships rockets, drugs and medicine etc. Polymers can be natural

or synthetic depending upon their source. The database of life called DNA is a protein

that regulates metabolism in a living body, is a natural polymer. Other kind of natural

polymers includes, wood, silk, cellulose, cotton, fur, etc. Ever since man has exposed the

fascinating properties and applications of polymers by changing their architecture,

functionality, size and tacticity. Moreover, the properties of natural polymers are

improved by modifications. Further developments resulted in synthesis of polymers other

than available naturally. In 1910, Bakelite was the first commercialized synthetic

polymer. About ten years later Staudinger postulated that polymers either natural or

synthetic are large molecules consisting of small units, covalently bonded to each other in

order to build the polymers with advance properties that greatly differ from their starting

material and primarily depends upon their size. Polymer is a Greek word meaning many

parts; ‗poly‘ for ‗many‘ and ‗mer‘ for ‗parts‘. Nowadays, polymer technology emerges on

a high flight, and synthetic polymer chemistry developed as an economical cheap

alternate for natural fibers having outstanding properties compared to conventional

materials like ceramics, wood and metals. Furthermore, synthetic polymers can be

tailored as per requirements of the final applications.

1.1 Properties of Polymers

Properties of polymers are classified on the basis of the scale on which the characteristic

is defined. It is mainly influenced by type of monomers and their arrangement within a

single chain of polymer such as random, statistical, block, etc. Basically, these structure

based properties of polymer define their overall physical characteristics. Furthermore,

chemical properties of the polymers also transform by the chains interaction due to

different physical forces.

3

1.1.1 Monomers and Repeating Units

Polymers consist of a similar repeat units are called homopolymers e.g. polybutadiene,

polystyrene, poly(2-vinylpyridine), polycarbonates etc., while the polymers having more

than one kind of repeating units are termed as copolymers. In copolymer at least two

types of structural units are present, therefore it can be of different types on the basis of

arrangement of repeating units along the chain. These include: alternating copolymer,

periodic copolymer, statistical copolymer and block copolymer Figure 1-1. Synthesis of

copolymers with unique architectures, morphology and composition is attaining a great

interest in scientific research because of their versatile applications in various field

(Speight, 2010).

Figure 1-1: Various kind of copolymers

4

The properties of polymers depend upon the number of repeating units in the polymer

chain denoted by a symbol ―n‖ and called as the degree of polymerization (DP) (Pasch,

2013).

1.1.1.1 Block Copolymer

Block copolymer is a distinctive kind of polymer in which each molecule comprises two

or more different segments of different monomers covalently bonded together in some

architecture. Block copolymers can be classified by the number of blocks and their

arrangement. Depending upon the number of blocks they are diblocks (AB), triblocks

(ABA), and multiblocks as shown in Figure 1-2.

Figure 1-2: Block copolymers having di-, tri- and multi-blocks

Similarly, according to arrangement, they can be linear, stars, brushes, rings, combs,

dumbbell-shaped, tree-shaped or H-shaped. Each block represents different physical and

chemical behavior due to which wide range of possible interesting properties can be

obtained by a single macromolecule. Various schematics arrangements of block

copolymers are presented in Figure 1-3.

5

Figure 1-3: Various schematic representations of shapes of block copolymers

1.1.1.2 Amphiphilic Block Copolymer

Additional treatments and modifications are often imperative for speciality applications

like tissue engineering, healthcare, nano-electronic devices, data storage materials, drug

delivery, alternative energy resources, cosmetics, etc. The prerequisite properties of

polymeric materials cannot be accomplished by standard polymers. In order to design a

smarter polymers or macromolecules with desirable sophisticated properties several

arrangements, modifications are required (Darling, 2007; Lutz, 2008). Among various

6

architectures of reported materials, amphiphilic block copolymers emerge as a new class

of polymers, exhibit multiple functionalities in a single polymer chain. The potential

applications of these amphiphilic assemblies are found in phase transfer catalysis, nano-

reservoirs, targeted drug delivery, gene therapy, metal nanoparticles, stabilization of non-

aqueous emulsion, etc. (Alexandridis, 1996; Kong, Li, Jin, Ding, & Shi, 2010; Riess,

2003; Riess & Labbe, 2004; Thurmond, Kowalewski, & Wooley, 1997; Wang, Winnik,

& Manners, 2007). It has a tendency to fabricate high-density arrays with desired

functionality in a predictable and controllable way to prepare materials with new and/or

improved physical properties for use in electronic storage devices, separation at

molecular level, screening of DNA and in combinatorial chemistry (B. J. Kim et al.,

2007; B. J. Kim, Bang, Hawker, & Kramer, 2006).

1.1.2 Microstructure

The microstructure or configuration of a polymer relates to the arrangement of monomers

within a polymer chain. The arrangement of monomers have a huge impact on the

polymer properties e.g., two different variety of natural rubbers having same monomers

may exhibit different durability because of the arrangement of these monomers.

1.1.2.1 Polymer architecture

Polymer properties are greatly influenced by their shape and architecture, e.g. branched

polymers have very different properties from their linear counterparts. Branched

polymers consist of side chains or branches on a backbone. Branched polymers can be

further classified as brush polymers, star polymers, dendronized polymers, comb

polymers, dendrimers, and ladder polymers etc.

1.1.2.2 Chain length

Properties of a polymer such as solubility, melting and boiling temperatures, and

viscosity are strongly reliant on polymer chain length. Furthermore, increasing chain

length leads to decreased mobility of a chain, high glass transition temperature (Tg), and

increase in the strength and toughness. This change in physical behavior is due to

increase in chain entanglements which internally enhance secondary interactions such as

7

Van der Waals interaction. These weak attractions bound the chains in a fix position and

minimize deformations, both at elevated temperatures and stresses.

1.1.3 Polymer Morphology

Polymer morphology explains three dimensional organization and ordering of polymer

chains at a micro level in space.

1.1.3.1 Crystallinity

Two different regions are found in synthetic polymers i.e. amorphous and crystalline

regions. Synthetic polymers are said to be crystalline when the polymer has region of

three-dimensional ordering at atomic scales. The degree of crystallinity is a volume

fraction or weight fraction of crystalline form. Degree of crystallinity is taken as zero for

non-crystalline polymers while one for completely crystalline polymers.

The appearance of polymers changes with the degree of crystallinity. Polymer will tend

to be transparent if degree of crystallinity approaches 0 to 1, while polymers will be

opaque with the value in between 0-1, because of light scattering by crystalline or glassy

regions.

1.1.3.2 Radius of gyration

The volume occupied by a polymer coil is usually denoted as radius of gyration (Rg). It

can be define as a mean distance from center of mass to any point of a polymer coil. It is

an experimental quantity that gives information about size of the polymer coil in solution.

Where,

Rg : Radius of gyration of polymer coil

rmean : mean position of monomers

N : number of polymer coils

8

1.1.4 Phase Behavior

1.1.4.1 Melting point

In polymer chemistry, melting point describes a temperature that causes change from a

crystalline to solid amorphous form. Melting temperature is related to thermoplastic polymers

only because thermosetting polymer crosslink irreversibly at elevated temperatures.

1.1.4.2 Glass transition temperature

It is the most important parameter in preparation of synthetic polymers. Tg is the

temperature at which amorphous region of the polymer converts from a viscous rubbery

form to a glassy brittle solid on cooling.

1.1.4.3 Mixing behavior

Generally, polymers are not miscible because the entropy of the polymer chains are far

less than small molecules. On the other hand, the energy of mixing depends on per

volume basis for small molecule and polymer. As the polymer chain increases, free

energy of mixing for polymer also increases that makes the solvation process less

favorable. Therefore, it is difficult to make a concentrated solution of polymers.

1.1.5 Chemical Properties

Chemical properties of the polymers are greatly affected by attractive forces between

polymer chains. The long chains of polymers amplify the interchain forces. Polymers

offer various physical interactions with their neighboring chains in a solution depending

upon different functionalities. These interactions include hydrogen bonding, ionic

bonding, dipole-dipole interactions etc. For instance, polymers consisting carbonyl or

amide groups form hydrogen bonds with other chains. Similarly, dipole-dipole

interactions exists amongst the carbonyl C=O oxygen and the H-C hydrogen in

polyesters. Polyethylene chains have weak vander waal forces.

9

1.2 Poly(2-vinyl pyridine)

Polymers containing pyridine moiety have widely been used as a capping agent or ligand

to stabilize the nanoparticles (Carotenuto, Pepe, & Nicolais, 2000; Lekesiz, Kayran, &

Hacaloglu, 2015; Shan & Tenhu, 2007; Walker, St. John, & Wisian-Neilson, 2001).

Among them poly(2-vinylpyridine) (P2VP) is the best candidate for chelation of metal

nanoparticles. The presence of nitrogen atoms in the pyridine moiety have a strong ability

to coordinate with metal ions or metallic nanoparticles through steric stabilization that

restrains the aggregation of the metal nanoparticles (Jang, Khan, Hawker, & Kramer,

2012; Lekesiz et al., 2015; Mössmer et al., 2000; Voulgaris, Tsitsilianis, Grayer,

Esselink, & Hadziioannou, 1999; Youk et al., 2002). Kunz et al. (Kunz, Shull, & Kellock,

1993) showed that the contact angle of P2VP is very low with Au (9°). PS-P2VP block

copolymer have been used to demonstrate precise control of the particles location within

the P2VP domain (Chiu, Kim, Kramer, & Pine, 2005). Gittins et al. (David I Gittins &

Frank Caruso, 2001) and Gandribert et al. (Gandubert & Lennox, 2005a) showed a

favorable interaction between pyridine and AuNPs surface using 4-(dimethylamino)

pyridine for the stabilization of AuNPs. P2VP forms random coil conformation in

solution which associates with the metal atoms and increases the probability of nucleus

formation (Carotenuto et al., 2000; Gandubert & Lennox, 2005c; Youk et al., 2002).

Several studies have been conducetd with P2VP based nanoparticles. P2VP based

amphiphilic block copolymers have received extensive attention in a field of

nanotechnology because they have the ability to self-assemble in particular solvent. They

form stable micelles at a nanoscale (i.e. 10 to100 nm) that provides an effective way for

controlling diverse range of 1D, 2D, 3D metallic nanoparticles patterns within a specific

location in polymer domain (Ikkala & ten Brinke, 2004; Quake & Scherer, 2000; Schmitt

et al., 1997; Xia et al., 1996). Preparation of metallic NPs in the block copolymer

template through micellization is a popular method. This method increases the stability of

nanoparticles that can be achieved easily and economically in terms of effectiveness and

efficiency (Shan & Tenhu, 2007; Torrisi, Ruffino, Licciardello, Grimaldi, & Marletta,

2011). An important role of copolymer systems interacting with colloidal metal

nanoparticles is to allow the initial small size to be maintained by preventing coagulation

10

and accurately control the placement of NPs within the block copolymer template.

Therefore, a profound understanding is required with regard to the interaction between

the particle surface, capping agent, and the polymeric matrix for controlling the 3D

structure of nanomaterials (B. J. Kim et al., 2006). Furthermore, the functional groups

and mechanism concerned in colloid stabilization differ through pendant groups attached

to the polymer backbone (e.g., pyrrolidone (Carotenuto, 2001), thiol (Shimmin, Schoch,

& Braun, 2004), or pyridine groups (Jang et al., 2012) etc.), which offers varying particle

size and stability (Badawy et al., 2010b; Ju-Nam & Lead, 2008; Walker et al., 2001).

1.3 Polymer-Metal Nanomaterials

Fabrication of polymer-metal nanomaterials is a potential route for synthesis of advanced

novel functional materials such as highly effective catalysts, band gap devices, chemical

and biochemical sensors, and secondary storage devices. For certain applications such as;

catalysis, optics and electronics, it is suitable to prepare stable, small but not fully

cavitated, therefore, active sites of particles are accessible, otherwise the efficiency of

nanoparticles (NPs) reduces. Two challenges that are imperative in this respects are; (1)

prevention of NPs from aggregation without jamming active surfaces on the nanoparticle

and (2) control over the size, shape, and size distribution of NPs. Various natural

macromolecules like proteins, flavonoids, liposomes and polysaccharides as well as

synthetic macromolecules such as polymers have been employed for construction of

nanosensors (Fang et al., 2011; Gandubert & Lennox, 2005a).

Polymers are especially suitable as template for the encapsulation of NPs because of their

fairly uniform composition and structure that help in the fabrication of well-defined NPs

and prevent agglomeration or segregation of nanoparticles (Aurélien et al., 2014;

Mössmer et al., 2000; Shan & Tenhu, 2007; Tyagi, Kushwaha, Kumar, & Aslam, 2011a;

Walker et al., 2001; Youk et al., 2002; Yu, Chien, & Chen, 2008). Encapsulated NPs are

stabilized by steric effects, thus a considerable fraction of NPs is unprotected and more

active surfaces of NPs are available for further use. The functional groups in the polymer

also control solubility of NPs and used as handles to link two surfaces and other polymers

(Crooks, Zhao, Sun, Chechik, & Yeung, 2001). Various research groups reported the

11

polymeric templates as a well beyond that of a simple casting mold (Abraham, Kim, &

Batt, 2007; Balazs, Emrick, & Russell, 2006; Bockstaller, Mickiewicz, & Thomas, 2005).

A variety of metallic nanoparticles including Cu, Ag, Au, Pt, Pd, Cr and Rh etc have been

synthesized with P2VP to control geometry, size and properties of these nanomaterials

(Aurélien et al., 2014; Jang et al., 2012; Sana Rahim, 2017; Yu et al., 2008). The template

is removed chemically or thermally if naked nanomaterial is required. The technique

provides monodisperse particles with a diversity of sizes, shapes, and chemical

compositions can be fabricated (Kang & Taton, 2005; B. J. Kim et al., 2006).

As a significance of their multiple functionalities and 3D structure, polymers are also able

to stabilize a number of ions and molecules. Stabilization mainly depends on the nature

of the particles, chemical composition and the cavity size of the polymers. Metal interacts

with polymers by the driving forces such as, covalent bond formation, secondary

interactions play a vital role such as complexation reactions, electrostatic interactions,

and various types of weaker forces (Vander Waals, hydrogen bonding, etc.), steric

confinement, and combinations thereof (Boal, Ilhan, DeRouchey, & Thurn-Albrecht,

2000; Caruso, Caruso, & Möhwald, 1998; J. Jin et al., 2001; J. Liu et al., 1999; Naka,

Itoh, & Chujo, 2003; Patil, Mayya, Pradhan, & Sastry, 1997).

Public and private sectors funded to the advancement of research in the field of

nanotechnology and its applications in other fields like molecular biology, surface

science, semiconductor physics, and organic chemistry. The nanotechnology based

research and their uses are diverse starting form preparation of commonly used physical

devices to innovative advances to established advanced materials with nano-dimensions.

Advancements in the fields such as biomaterials, medicine and electronics etc are very

much related to the progress in the field of nanotechnology. Additionally,

nanotechnology nurtures about toxicity and environmental impact of nano-materials on

the world economy (Bockstaller et al., 2005; Jiang, Oberdörster, & Biswas, 2009b; H.-C.

Kim, Park, & Hinsberg, 2009; Toshima & Yonezawa, 1998).

12

1.4 Nanoparticles

Nanoparticles (NPs) are achieving excessive interest since it establishes a connection

between atomic structures and its bulk material. The material properties are influenced by

the size of material approaches the nano-level and as the surface area per volume of a

material is enhanced. The exciting and unpredicted properties of NPs are realized due to

increased surface area of the material. For specific applications, properties of

nanoparticles such as size, shape, size distribution and surface characteristics are tuned

accordingly. Nanoparticles exists in different shapes as shown in Figure 1-4.

Figure 1-4: Various shapes of different nanoparticles

1.4.1 Characteristics and Applications of Nanoparticles

Various nanosystems are used according to their potential for different applications

(Nahar et al., 2006). The characteristics and applications of some nanosystems are

summarized in Table 1.1.

.

13

Table 1-1: Different characteristics and applications of nanoparticles

Types of Nano-

systems Size (nm) Characteristics Applications

Carbon nanotubes

(CNTs)- single

walled nanotube,

SWNT) or multiple

layer (multi-walled

nanotube, MWNT).

0.5–3

diameter

Remarkable strength,

distinctive electrical

properties including

conducting, semi

conducting, or insulating

Enhanced solubility due

to functionalization,

increase penetration to

cell cytoplasm and

nucleus, used as carrier

for gene and peptide

delivery

Dendrimer Less than

10

Highly branched

polymer; having three

major parts surface, core

and branch

Long circulation,

controlled and targeted

drug delivery, liver

targeting

Liposome 50–100

Phospholipid vesicles,

biocompatible, high

entrapment efficiency

Long circulation, active

and passive gene delivery

Metallic

nanoparticles

Less than

100

Au and Ag colloids,

much smaller in size,

enhanced surface area to

volume of a particle,

stable

Highly sensitive

diagnostic assays,

thermal ablation, drug

and gene delivery and

enhanced radiotherapy

Nanocrystals

Quantum dots 2–9.5

Semi conducting

material; Size 10-100 Å,

high photo stability,

bright fluorescence with

narrow emissions, long

range UV excitation,

Imaging of liver cell,

immunoassay, DNA

hybridization

Polymer micelles 10–100

Amphiphilic micelles,

enhanced drug loading

efficiency, biostability

Active and passive drug

delivery

Polymeric

nanoparticles 10–1000

Highly functional, small

quantity offers complete

drug protection

Excellent candidate for

controlled drug delivery,

surface modified NPs

used as active and

passive drug delivery and

chemosensors

1.4.2 Quantum Confinement Effects

According to the quantum mechanical rules (Aharonov & Bohm, 1959), NPs having a

diameter of 1-100 nm displays the quantum confinement effect when particle size is too

small comparable to the wavelength of the electron. The confinement means to restrict

14

the random motion of electrons in a confined space having a specific energy, causes a

transition from continuous to discrete energy levels and the word quantum defines the

atomic realm of particles. Therefore, as particle size decreases up to a nano level, the

difference between the two energy level of confining dimension becomes discrete and

ultimately the band gap and energy of the band gap increases.

Physical properties of the NPs be strongly subjected to particle size, shape, interparticle

distance and nature of the protecting agent. The electrons on the surface of particle show

tunneling effect when combined with their neighboring particles. The tunneling process is

an effect that differentiate intra and intermolecular processes that can be detected by

impedance measurements (Lambe & Jaklevic, 1968). The quantum size effect is

pronounced if the particle size is in the range of de Broglie wavelength of valence

electrons. Quantum-mechanical rule explained that freely moveable electrons in 0D

quantum dots display particular cumulative oscillation frequency of plasma resonance

that leads to characteristic plasmon resonance band (PRB). For example, the

characteristics band of gold nanoparticles is observed around 500-600 nm (Daniel &

Astruc, 2004), while the silver nanoparticles show a characteristics band between 350-

450 nm (Mulfinger et al., 2007; Solomon et al., 2007). Unlike bulk materials, a gap

between the conduction and valence band is generated in NPs which cause size

dependent quantization. NPs diameter around 20 nm induces standing electronic waves

that create discrete energy levels and cause large number of differences in optical and

electrical properties of nanoparticles. This kind of flexibility is required for number of

potential applications such as electrometers, transistors, switches, oscillators, catalysis,

and biosensors (Boisselier & Astruc, 2009; Daniel & Astruc, 2004; Saha, Agasti, Kim,

Li, & Rotello, 2012).

1.4.3 Surface Plasmon Resonance (SPR)

SPR has gained considerable attention especially in the field of catalysis and

optoelectronics due to their optical properties as described by Mie theory. According to

the Mie theory (Fu & Sun, 2001) the overall sum of electromagnetic oscillations is

directly proportional to surface plasmon absorption and scattering by the particles. It

15

relates the surface plasmon band of spherical NPs with dipole oscillations of free

electrons in conduction band that occupies energy state above the Fermi energy.

The change in surface plasmon resonance band (SPR) is observed with decreasing the

core size and shape of the NPs. This decrease in size dominates the quantum size effect

that causes blue shift and spectral transitions. SPR bandwidth and absorption maximum

(Amax) are also affected by temperature, dielectric constant of the medium and refractive

index of solvent. The presence of a ligand or capping agent changes the refractive index

which causes either red or blue shift, a deviation from Mie theory. The Mie theory deals

with the bare nanoparticles, nonetheless, ligand conjugated NPs show deviation from

Mie theory (Ateeq et al., 2015; Daniel & Astruc, 2004).

The shift in SPR band is significant when nitrogen and sulphur containing compounds are

used as ligands (Daniel & Astruc, 2004; Sana Rahim, 2017). These ligands strongly

interact with electronic cloud on the surface of the particles. Therefore, SPR does not

always follow the Mie theory. Non-spherical NPs show a red shift because spacing

between NPs reduces as the gap between conduction and valence band decreases. This

ability of NPs makes them alluring candidates in optical measurements, e.g. impurities

are also detectable as the refractive index of MNPs changes compared to that of their

oxides and chlorides. Temperature also affects the SPR band that can be explained by

electrons dephasing mechanisms. In this mechanism electron-electron interactions are

observed instead of electron photon coupling. Due to enhanced sensitivity of SPR

position, these NPs are applied for biosensor and chemosensors applications (Ateeq et al.,

2015; Chah, Hammond, & Zare, 2005; Daniel & Astruc, 2004; Sana Rahim, 2017).

1.5 Classification of Nanoparticles

Nanoparticles can be classified into different classes such as 0-, 1-, 2- and 3-dimensional

structures, Figure 1-5.

1.5.1 Zero dimensional nanoparticles

Zero dimensional nanoparticles (0D) includes clusters.

16

1.5.2 One dimension nanoparticles

One dimensional (1D) system includes nanotubes, fibers and rods.

1.5.3 Two dimension nanoparticles

Two dimensional system (2D) includes films and coats. These thin films or coats are

commonly used in solar cells, chemical and biological sensors, storage devices, magnetic

and optical devices etc.

1.5.4 Three dimension nanoparticles

Three dimensional system (3D) includes quantum dots (QDs), dendrimers, and fullerenes

(Carbon 60).

Figure 1-5: Various kind of nanoparticles

1.6 Metallic Nanoparticles

The term metallic nanoparticle (MNPs) is used to explain nanosized metals having

dimensions in the size range of 1‐100 nm. In 1857 Micheal Faraday recognized the

existence of MNPs in solution for the first time and in 1908 Mie quantitatively explained

metallic nanoparticles on the basis of their colour, so called Mie theory. MNPs offer high

surface area to volume ratio compared to their bulk materials, larger surface energies,

quantum confinement, short range ordering, specific plasmon excitation and subsequently

unique chemical properties.

17

Metallic nanoparticles such as, noble metals e.g. Au (Boisselier & Astruc, 2009) , Ag

(Podsiadlo et al., 2005; Robinson et al., 2008), Pd (Ung et al., 2009), semiconductors e.g.

ZnS, CdS, CdSe (Bawendi, Sundar, & Mikulec, 2007; Boisselier & Astruc, 2009), TiO2

(Drbohlavova, Adam, Kizek, & Hubalek, 2009), InP and PbS (Rogach, Eychmüller,

Hickey, & Kershaw, 2007), Si (O‘Farrell, Houlton, & Horrocks, 2006) can be constituted

in various materials. The formation of MNPs is realized by reducing salts of the metals

with reducing agents (Sun & Zeng, 2002) in the presence of a stabilizer like polymers,

dendrimers, microgels, surfactants, and colloids (Abraham et al., 2007; Daniel & Astruc,

2004; Gandubert & Lennox, 2005c; Jaramillo, Baeck, Cuenya, & McFarland, 2003;

Perrault & Chan, 2009; Shan & Tenhu, 2007; Youk et al., 2002). Polymer provides

enhanced surface area/volume ratio of NPs that resulted in higher reactivity and offers

stability to NPs through steric or electrostatic repulsion (H.-C. Kim et al., 2009).

Currently, metal and metal oxide NPs are extensively studied because of their wide range

of applications in optics, catalysis, photophysics, and medicinal sciences e.g. imaging,

sensing, photodynamic therapy, hyperthermia, and drug delivery. In various circumstances,

chemically inert metal nanoparticles are required in order to reduce toxicity and other side

effects. Recently, it is observed that metallic nanoparticles increase the potency of some

drug-molecules e.g., Kotov‘s (Podsiadlo et al., 2008), studied that 6-mercaptopurine

stabilized gold nanoparticles kill leukemia cells more efficiently than 6-mercaptopurine. Jin

and He (T. Jin & He, 2011), reported that the antibacterial potential of nisin is enhanced

noticeably when nisin was used with magnesium oxide (MgO) against E.coli in the culture

of food. Recently, AuNPs and AgNPs, have been investigated extensively because of their

exceptional optical, catalytical and electrical properties (Abraham et al., 2007; Daniel &

Astruc, 2004; Gandubert & Lennox, 2005c; Perrault & Chan, 2009; Schaaff & Whetten,

2000; Shan & Tenhu, 2007; Toshima & Yonezawa, 1998; Youk et al., 2002). AuNPs are

commonly used in drug/gene delivery and photothermal therapy. Moreover, AgNPs are

used as antibacterial agents while Fe2O3-NPs are used in hyperthermia and magnetic

resonance imaging (MRI). Chemically reactive species of MNPs are rarely used.

18

1.6.1 Gold Nanoparticles (AuNPs)

Although gold is the subject of investigations in science since ancient times, its

revitalization now leads to exponential growth of publications in the emerging fields of

nanotechnology and nanoscience. Among all nanoparticles, AuNPs are the stable metal

nanoparticles and are used in various fields such as material sciences, biology and catalysis

because of their captivating features that include individual particles behavior, quantum

size effect such as size-dependent optoelectronic and magnetic properties etc. Gold is an

efficient electron conductor. Due to its potentials in these fields and in the bottom-up

approach of nanotechnology, it is considered to be a key material and building block in the

21st century (Daniel & Astruc, 2004; Pooja, Panyaram, Kulhari, Rachamalla, & Sistla,

2014; Shan & Tenhu, 2007).

AuNPs are synthesized by reduction of gold salts, Au (III) salts are mostly used. In 1951,

Turkevitch (Turkevich, Stevenson, & Hillier, 1951) reduced HAuCl4 salt of gold using

citrate in water. Controlled nucleation of gold particles to attain the monodispersity of

particle size in gold suspension was also performed (Frens, 1973). Brust-Schiffrin

introduced two phase synthesis using thiol ligand for the stabilization of gold particles in a

liquid-liquid system (Brust, Walker, Bethell, Schiffrin, & Whyman, 1994). Jadzinsky et al.

investigated the characteristics of AuNPs structure by using X-Rays diffraction (Ackerson,

Jadzinsky, & Kornberg, 2005; Jadzinsky, Calero, Ackerson, Bushnell, & Kornberg, 2007).

Haruta et al. (Haruta, 1997; Haruta & Daté, 2001; Haruta, Kobayashi, Sano, & Yamada,

1987; Haruta et al., 1993; Haruta, Yamada, Kobayashi, & Iijima, 1989) used AuNPs coated

Co3O4, TiO2, or Fe2O3 as a catalysts for CO2 hydrogenation, catalytic combustion of

methanol, H2 and CO oxidation, water gas shift reaction and NO reduction. Gold

nanoparticles were also used as rectifier for microchannels in chip-based capillary-

electrophoresis devices (Daniel & Astruc, 2004). Encapsulation of AuNPs prevailed over

photo oxidation in commercial devices (Xue et al., 2014). Gold nanoparticles are used to

study the structures, morphology, properties, and applications of biological, inorganic and

molecular nanomaterials (Bindhu & Umadevi; Jang et al., 2012; Kang & Taton, 2005; Saha

et al., 2012; Sohn & Seo, 2001; Spatz, Mößmer, & Möller, 1996).

19

1.6.2 Silver Nanoparticles (AgNPs)

Silver is another commonly used metal for fabrication of nanoparticles. Silver nanoparticles

(AgNPs) are synthesized by various physical and chemical protocols. The physical

procedures involves evaporation or condensation techniques and by using laser. While,

chemical methods include the reduction of silver ions into silver metal from silver salts (El-

Nour, Eftaiha, Al-Warthan, & Ammar, 2010).

Silver nanoparticles (AgNPs) have been used as an antibacterial agents for past decades.

Nowadays, AgNPs are used in textile industry and in commercial detergents to kill bacteria

and to prevent the spread of bacterial diseases. Conjugation of various biocides to AgNPs

enhances antibacterial activity. Capped nanoparticles has the ability to bind with two

different biocides, inorganic (silver nanoparticles) and organic (capping agents), which

offers different metabolic pathways to target the organism more effectively inside the body

(Chernousova & Epple, 2013; J. S. Kim et al., 2007; Rai, Yadav, & Gade, 2009).

1.6.3 Other Nanoparticles

Other metallic nanoparticles include zinc (Hattori, Mukasa, Toyota, Inoue, & Nomura,

2011), palladium (Corthey et al., 2012), Copper (Ruparelia, Chatterjee, Duttagupta, &

Mukherji, 2008) etc. Some metal oxide nanoparticles such as Fe (III) oxide (Rosen, Chan,

Shieh, & Gu, 2012), titanium oxide (Shiraishi, Ikeda, Tsukamoto, Tanaka, & Hirai, 2011),

zinc oxide (Meulenkamp, 1998) etc. are also used. Certain rare earth doped nanoparticles

have also been employed in the field of nanotechnology (Bouzigues, Gacoin, &

Alexandrou, 2011).

1.7 Applications of Metallic Nanoparticles in Various Fields

The quantum size effect and high absorption coefficient related to excitation of surface

plasmon of MNPs opens a door for a broad range of applications in many fields. The

probability to manipulate, amplify and concentrate light through surface plasmon at the

nanoscale offers to improve optical properties in a controlled way. Myriad of potential

applications of surface plasmon in various fields are reported in literature.

20

1.7.1 Biomedicines

NPs have very small size, comparable to biological objects such as viruses, DNA

molecules, bacteria and other cells. Therefore, it is possible to use NPs to treat these

microorganism by interacting individually and increase efficiency and specificity of

medical treatments. Moreover, gold and silver NPs are highly biocompatible and easily

functionalize with thiol, nitrogen and oxygen containing organic molecules. Therefore, in

vitro applications are well established.

1.7.1.1 Drug delivery

NPs are useful for the delivery of drugs that induce undesired effects on normal tissues.

Controlled drug delivery overcome these secondary effects. For the purpose, the surface of

the drug is covered with NPs that avoids interaction with non-targeted cells. As the drug

reaches the targeted cells, the coating is removed. It also helps to control the release rate

and improvement in the effectiveness of the drug.

1.7.2 Energy

The efficiency of photovoltaic cells can be increased by 10-15% by incorporating Ag and

Au NPs on the cell surface. The efficiency of electrical devices is highly dependent upon

size, shape and spatial distribution of the NPs because interacting effects of light scattering

and absorption processes are different with variations in above-mentioned parameters.

1.7.3 Environment

1.7.3.1 Catalysis

Metallic NPs also exhibit catalytic activity that can be improved upon light illumination to

excite SP. The excited electrons participate in the oxidation of the products that are adsorbed

on the catalytic surface. The catalytic surface efficiency will depend critically on the light

absorption process that in turn depends on the number of electrons or holes in the material.

1.7.3.2 Chemosensing

Chemosensor is defined as a molecule which generates a response in the presence of

21

chemical stimulus. Recently, extensive attention has been given to the molecular design of

fluorescent and/or colorimetric chemosensor. One or more macroscopic photophysical

properties (e.g. color and strength of fluorescence and UV–Vis absorption) alters with the

change in molecular design by addition of a target species (Z. Li & Zhang, 2006; Umali &

Anslyn, 2010). AgNPs are used to measure the modification in single nucleotide of DNA.

Dark DNA-Ag clusters were used for the detection of guanine rich sequence of DNA.

Silver nanoclusters are used for the specific detection of cysteine in presence of other

amino acids. Our group used T-lymphocytes with pyridinium thioacetate capped AgNPs

detection of copper in real samples (Anwar, Shah, Muhammad, Afridi, & Ali, 2016).

A major challenge affecting the development of chemosensing protocol is the fabrication of

sensing elements that specifically distinguish analyte molecule in a group of structurally

similar molecules. The popularity of a design system is determined by the comparative ease

by which it is improved to many applications.

Various techniques including UV-visible spectroscopy, fluorescence spectroscopy,

voltammetry and FTIR are used to determine the efficiency of NPs as a sensor.

1.8 Synthetic Approaches of Nanoparticles

Nanotechnology manipulates matter at atomic or molecular level in which at least one

dimension is above 1 nm and below 100 nm because quantum statistical and mechanical

effects of a system becomes more pronounced and significant at the size of 100 nm. All the

physical properties such as mechanical, electrical, and optical properties are different from

the macroscopic systems due to quantum size effect at nanoscale e.g., stable materials like

aluminum becomes combustible; insoluble gold becomes soluble; opaque copper converts

to transparent and inert gold becomes a robust catalyst. Nanoparticles can be synthesized

by the following approaches

Top down approach

Bottom up approach

22

1.8.1 Top-down approach

The top-down approach means starting from larger particles (top) and reduce them to

smaller particles (bottom). In this approach, large pieces of material break down to generate

the required smaller nanostructures, Figure 1-6.

1.8.2 Bottom-up approach

The ―bottom-up‖ approach means smaller (bottom) to larger (up). In this approach, small

pieces (atoms or molecules) assembled together and form a desired larger nanostructure,

Figure 1-6.

Figure 1-6: Synthetic approaches of nanoparticles

23

1.9 Limitations in Nanoscale Approaches

The versatile applications and facile synthesis of NPs made them a fast growing area of

research. Although, the synthesis of NPs is simple, nonetheless, there are limitations in

context of selectivity and precision the size. For examples, control over nuclearity can be

achieved by various methods in the synthesis of clusters, however, nanoclusters with high

monodispersity in bulk quantities is still a subject of research.

1.10 Characterization of Nanoparticles

Nanoparticles characterization is mainly based on their size, surface charge and

morphology using innovative microscopic practices such as scanning electron

microscopy (SEM), and transmission electron microscopy (TEM). Physical stability of

NPs depends upon the mean particle diameter and size distribution of NPs. The features

like redispersibility and physical stability of the polymer dispersion are influenced by the

surface charge of NPs. Various characterization techniques for size, size distribution,

morphology, stability, surface charge of NPs and other factors related to NPs such as

drug interactions, drug loading and releasing efficiency and drug stability are summarized

in Table.

Table 1-2: Different characterization techniques for various parameters related to

nanoparticles

Parameters Characterization Techniques

Carrier-drug interaction UV-Visible spectroscopy, FTIR, Zetasizer

Surface charge distribution Zeta potentiometer

Drug loading/releasing UV-Visible spectroscopy

Fluorescence spectroscopy

Surface morphology AFM, SEM, TEM

NPs stability UV-Vis spectroscopy, DLS, Zetasizer

Size and size distribution of NPs DLS, Zetasizer, AFM, SEM, TEM

24

1.10.1 Particle Size and surface morphology

Nanoparticles are mainly assessed by particle size and size distribution as well as their

surface morphology. Atomic force microscopy reveals detailed information with regard

to the morphology, and the particle size and its distribution. Particle size of NPs has

profound effect on the drug loading efficiency and their sensing performance.

Subsequently, small size nanoparticles tend to agglomerate with time. Therefore, a

mutual compromise between small size of NPs and maximum stability is always sought

(Judefeind & de Villiers, 2009). New advances in analytical techniques for the

elucidation of NPs size and surface morphology are discussed below.

1.10.1.1 Light scattering methods

John Tyndall studied the light scattering phenomena in solution containing different sized

particles in 1869. He observed that different sized particles scattered light in different

ways. Later in 1871, Lord Rayleigh proposed a theory of light scattering in which he

stated that, ―In light scattering, light is scattered in the form of propagating energy and it

deflect from a straight path by irregularities in the medium exist‖, Figure 1-7 (L. Mei,

Somesfalean, & Svanberg, 2014).

Figure 1-7: Process of light scattering in solution

25

1.10.1.1.1 Dynamic Light Scattering (DLS)

DLS studies a wide range of phenomena concerning the dynamical behavior of fluids

near critical points and determines size and radius of small particles in a solution

(Goldburg, 1999; Sartor). It measures fluctuation in time of the intensity or spectral

distribution arise from dynamical properties of macromolecules (Pecora, 1979). A

monochromatic light is passed through a solution that induces a Doppler Shift in the

particles present inside the solution having Brownian motion and change the wavelength

of incident light. This change in wavelength of light is directly proportional to the size of

the particles. The advantages of this method include automatized procedure, no

requirement of extensive experience and short duration of experiment. It allows

distinguishing polymer as monomer or dimer with different molar masses in small

amounts. Moreover, DLS offers measurements of molar mass, diffusion constant, radius

of gyration and several other parameters. However, the limitation of method is its less

accuracy for oligomers and polydispersed systems. Non-rigid macromolecules are

difficult to analyze because above the zero degree Kelvin molecules deviate from their

average position (Sartor).

1.10.1.1.2 Static Light Scattering (SLS)

SLS characterize the average molecular weight (Mw) of a large molecules such as

polymer or protein in solution by measuring the scattering intensity of light at various

angles that permits calculation of the root mean square radius or radius of gyration (Rg).

1.10.1.2 Scanning Electron Microscopy (SEM)

SEM offers numerous advantages in morphological and sizing analysis of NPs. It

determines the surface morphology, shape and size of the NPs by direct visualization.

However, it provides limited information with regard to average size distribution and true

population. During SEM characterization, solution of NPs is dried initially, mounted on a

sample holder and coated with a conductive metal (e.g. gold) with the help of a sputter

coater. Analysis of sample is done by scanning with a focused fine beam of electrons

(Pal, Jana, Manna, Mohanta, & Manavalan, 2011). Surface characteristics of the sample

26

is evaluated by secondary electrons emission from surface of the sample. Polymer

coating on the surface of NPs can often be damaged by electron beam therefore stability

of the polymer to withstand the electron beam is imperative.

1.10.1.3 Transmission Electron Microscopy (TEM)

The small size of nanostructures limits the application of conventional methods to

measure their physical properties. Transmission electron microscopy (TEM) offers

imaging, diffraction and spectroscopic information with an atomic spatial resolution of

the specimen. In TEM, sample preparation method is time consuming and difficult

because ultrathin film is required for the electron transmittance. When nano diffraction

atomic resolution electron energy-loss spectroscopy and nanometer resolution X-ray

energy dispersive spectroscopy methods are combined with high-resolution TEM

imaging, it offers critical basic studies of importance to nanotechnology. In TEM

characterization, nanoparticles solution is casted onto support films or grids and are fixed

either by a negative staining material such as uranyl acetate, phosphotungstic acid etc., or

by plastic embedding. It makes NPs to tolerate against the vacuum in the instrument and

ease of handling. Alternatively, NPs sample is also exposed to liquid nitrogen after

inserting in crystal ice. The surface characteristics of the sample are obtained by

transmission of a beam of electrons through an ultrathin film of sample (Jores et al.,

2004; Molpeceres, Aberturas, & Guzman, 2000).

1.10.1.4 Atomic Force Microscopy (AFM)

AFM is the tool of choice to build the relationship between structure and property at the

nanoscale level. AFM analysis is a critical step for investigating and manipulating the

fundamentals of macromolecules and the corresponding functions and applications. AFM

has been applied as a nanotechnology tool since it was invented in 1986.

AFM is known as ―Eye of Nanotechnology‖ and referred to as Scanning probe

microscopy (SPM). It is a high resolution imaging technique that offers ability to image

variety of surfaces characterized at the atomic level in the real space. It reveals

spatial resolution of individual surfaces of atoms and molecules which gives unique

27

perspective for scientific technology. Ultimately it makes possible to conceptually

study single molecule chemistry (Atwood, 2009; Blanchard, 1996; Magonov et al., 1991).

It has much broader potential to image any conducting or non-conducting surface that

enhance their applications in the field of nanotechnology. AFM offers the ability to grasp

happening at atomic or molecular levels and lead to discoveries in other fields like life

science, polymer science, electrochemistry, nanotechnology, materials science,

biotechnology and biophysics.

AFM measures interactive forces as a function of distance between the sample and the

tip. This force and distance relationship is denoted by a force-distance curve. Usually, tip

is sharp and 3-6 um tall pyramid with end radius of 15-40 nm (Figure 1-8). AFM lateral

resolution is about ~30 nm and the vertical resolution is up to 0.1nm.

Figure 1-8: Schematic representation of basic principle of AFM

AFM can operate at ambient conditions and performed in three different modes, which

can be applied for achieving different purposes depending on the properties of sample

and final target.

28

1.10.1.4.1 Contact Mode

The tip drags on the surface of the sample and the curves on the surface are analyzed

either directly by deflection of the cantilever or the feedback signal that keep the

cantilever at a fix position because static signals are prone to drift and noise, Figure 1-9.

Cantilevers having a low spring constant, k are used to achieve enough deflection signals

while keeping the interaction force low. Near the sample surface attractive forces are

quite strong that cause the tip to snap-in to the surface. Consequently, this mode is used at

a depth where the overall force is repulsive.

Figure 1-9: Contact mode of AFM

1.10.1.4.2 Tapping Mode

Contact mode is not suitable for the liquid samples at ambient conditions because the tip

sticks to the surface. Tapping mode is developed in order to overcome this problem. In

this mode, the tip is close to the sample surface up to short-range forces to be detectable,

Figure 1-10. Nowadays, it is frequently used AFM mode for the liquid samples or the

samples need to be operate at ambient conditions. The phase images are recorded in

tapping mode that gives information about the morphology and size distribution of the

sample. Sample contains different adhesion regions of different properties and varying

stiffness generate a contrast that is not visible in the topographical images.

29

Figure 1-10: Tapping mode of AFM

1.10.1.4.3 Non-contact Mode

The tip is not contacted with surface of the sample but the cantilever is oscillated at

its resonating frequency in non-contact mode. The van der Waals forces or any other long

range forces present above the surface of the sample which are strongest from 1 nm to

10 nm acts to decrease the cantilever resonating frequency. This decrease in resonating

frequency with the feedback loop system keeps an oscillation amplitude constant by

adjusting the average distance between tip and sample and allows to construct

topographical images of sample surface, Figure 1-11.

Figure 1-11: Non-contact mode of AFM

AFM offers a three-dimensional surface profile without any requirement of special

treatments that would change or damage the sample surface. AFM provides a platform to

work under ambient conditions, with liquid or solid sample even study living organisms

and other biological molecules with high resolution. But major limitation is that

30

knowledge obtained about the interior composition is not sufficient because surfaces are

less organized than interiors (charles E. Carraher, 2012). AFM probes normally cannot

measure overhangs or steep walls. To measure sidewalls, special AFMs and cantilevers

are prerequisite that are more expensive, have lower lateral resolution and other

additional artifacts.

1.10.2 Surface Charge

Surface charge determines the physical, chemical and biological interactions of NPs with

their environment. Zeta potential (Zp) also provides information about the stability of the

nanoparticles. Zeta potential indirectly measures the surface charge. It is evaluated by

measuring the potential difference between the surface of shear and the outer Helmholtz

plane. Consequently, zeta potential of NPs dispersions promote direct assessment of their

stability. High Zp values (positive or negative) show better stability and prevent particle

aggregation. Zp values are utilized in estimating nature and surface hydrophobicity of the

encapsulated material coated onto the surface or within the nanocapsules (Bhatia, 2016;

Dadwal, Solan, & Pradesh, 2014).

1.10.3 Drug Loading/Releasing

In order to determine the extent of the drug loading/releasing such information like how

much drug is encapsulated in nanoparticles is required. Drug loading/releasing efficiency

of the NPs is defined as; ―the amount of drug attached/released per mass of NPs or the

moles of drug per mg of NPs or mg drug per mg NPs (Kreuter, 1983; Magenheim, Levy,

& Benita, 1993).‖

Different techniques including high performance liquid chromatography (HPLC) and

UV-visible spectroscopy are used to determine drug loading/releasing efficiency.

1.10.3.1 High-Performance Liquid Chromatography (HPLC)

High performance liquid chromatography is a leading analytical technique used for

separation, identification and quantification of components in a complex organic mixture

e.g. polymers, biopolymers, and drugs etc. It is versatile and the most sensitive technique

31

that allows better separation in a short time by utilizing small amount of sample compared

to other liquid chromatographic methods. Chromatographic separation based on specific

interactions between sample molecules with the stationary and mobile phases provide an

additional variable for controlling and improving separation (Trathnigg, 1995).

1.10.3.2 UV-Visible Spectroscopy

UV-Vis spectroscopy is an analytical technique used for the quantification of various

analytes. It is an absorption spectroscopy in which analyte molecule absorbs

electromagnetic radiations in the ultraviolet-visible region (Förster, 2004; Macheroux,

1999). Molecules containing π or non-bonding electrons including transition metal

complexes, conjugated organic molecules, absorb energy to excite these electrons to anti-

bonding molecular orbitals. The more energy required to excite the electrons, the shorter

wavelength of light will be absorbed and vice versa.

UV-Vis spectrophotometer is used to measure the light absorbed by an analyte in UV-visible

range. A monochromatic light is passed through a sample containing analyte, and the

intensity (Io) is measured in comparison to the intensity of incident light (I). The

transmittance is calculated by a ratio of Io / I, and expressed in a percentage, %T, Figure 1-12.

Figure 1-12: Schematic representation of basic instrumentation of UV-visible

spectrophotometer

32

This method based on the principle of Beer-Lambert law for the quantitative

determination of concentrations of an absorbing species in solution.

According to the Lambert Beer law, absorbance is directly proportional to the

concentration of the absorbing species, c and to the path length, l of the cell, as denoted

by the equation,

Where,

A : Measured absorbance of the absorbing specie

I : Intensity of incident light at a particular wavelength

Io : Transmitted intensity

L : Path length of cell containing absorbing specie

C : Concentration of absorbing species

ε : Extinction coefficient or molar absorptivity

1.10.3.3 Fluorescence spectroscopy

Fluorescence spectroscopy is an analytical tool that analyzes fluorescence from a sample.

It contains a beam of light (i.e. ultraviolet light of electromagnetic radiations), that excites

the electrons in molecules of a compound and causes them to emit light in visible or UV

range.

Two types of instruments are used to detect the light emitted by the molecule; (1)

fluorometer in which filters are used to isolate the incident light and fluorescent light, and

(2) spectrofluorometer in which diffraction grating monochromators are used to isolate

the incident light and fluorescent light.

33

Principally, a light from an excitation source strikes the sample after passed through a

filter or monochromator. The sample absorbs a portion of the incident light that is emitted

then as fluorescent light that spread in all directions. A part of this fluorescent light

passes through a second filter or monochromator and strikes a detector, located at the

angle of 90° with incident light beam to minimize the risk of reflected or transmitted

incident light approaching the detector, Figure 1-13 (Moerner & Fromm, 2003).

Unlike UV-visible spectroscopy, independent spectra cannot be achieved easily. Various

aspects and affects can mislead the spectra; therefore, corrections are necessary to obtain

machine-independent spectra. Several kinds of distortions related to sample or instrument

related are discussed. In fluorescence spectroscopy, the light source intensity and

wavelength characteristics changes as the time passes during and between the

experiments. Moreover, there is no light source that has continuously same intensity

therefore, a beam splitter is placed next to excitation monochromator or filter to point a

fraction of light to a reference detector. In addition, filters or monochromators

transmission efficiency may change with the passage of time. The transmission efficiency

of monochromator also differs as the wavelength changes. An optional reference detector

is placed after the excitation filter or monochromator to overcome this problem (Weiss,

1999).

In fluorescence spectroscopy, it is necessary to use a cuvette made up of quartz. It

transmits wavelengths from 200 nm to 2500 nm, high quality quartz transmits up to

3500 nm. Other materials mask fluorescence due to sample.

Correction of all these instrumental factors is a tedious process. Nevertheless, It is

necessary to make corrections in case of measuring the quantum yield or to find the

wavelength with the highest emission intensity (Moerner & Fromm, 2003).

34

Figure 1-13: Schematic representation of basic principle of fluorescence spectroscopy

1.10.3.4 Fourier Transforms Infrared Spectroscopy (FTIR)

FTIR is an effective analytical tool for the determination of functional groups in a solid,

liquid or gaseous sample. It also gives information about structure, chemical composition,

bonding arrangement between constituents at the molecular scale (charles E. Carraher,

2012). FTIR spectrometer can operate in transmission or reflection modes. The

transmission mode is applicable for quantitative analysis and reflection mode is used for

the characterization of molecules that are not soluble at room temperature. FTIR

spectrometer instantaneously offers high-spectral-resolution data over a broad spectral

range. FTIR is used in all applications, their improved speed and sensitivity have

unfastened different areas of application. FTIR has applications in chemistry, geology

and biology and material science. Moreover, a variability of instrumental and sampling

configurations of IR spectroscopy makes it a multipurpose characterization method for

measurement of structure-property relationship of complicated systems under different

environmental conditions (Bhargava, Wang, & Koenig, 2003; charles E. Carraher, 2012).

35

1.10.3.5 Voltammetry

A set of electro-analytical techniques used in analytical chemistry for the detection of

analyte to be electroactive in nature. Voltammetry, gives information related

to analyte by measuring the current change with applied potential. It provides the

analytical data in the form of a voltammogram, which is obtained by plotting the current

produced by an analyte versus the potential of the working electrode (Compton & Banks,

2007).

Recently, modern three electrode system is employed that contains a reference electrode

(Re), an auxillary electrode (Ae) and a working electrode (We). Working electrode at a

desired controlled potential is in contact with analyte making easy charge transfer to and

from the analyte. While a Re has a known reduction potential. Ae is used as a helper with

We that passes all the current to balance the current on working electrode.

Various voltammetric techniques are used for the qualitative and quantitative detection of

analyte. Among them cyclic voltammetry (CV) is one of the potentio-dynamic

electrochemical technique to measure electrochemical properties of an electroactive

species in solution. In CV, the working electrode potential (Wep) is ramped linearly vs

time. After the set potential is reached, the Wep is ramped reversibly to attain the initial

potential. These potential ramp cycles may be repeated several times as needed. To

obtain a cyclic voltammogram, current applied at working electrode is plotted vs the

applied voltage. The effectiveness of CV is reliant on an analyte under study. The analyte

should be redox active within the applied potential window (Kissinger & Heineman,

1983; Laviron, 1974; Nicholson, 1965).

36

Chapter 2

Evaluation of morphology, aggregation pattern and size

dependent drug loading efficiency of gold nanoparticles

stabilized with poly (2-vinyl pyridine)

37

Abstract

Presence of basic nitrogen throughout the chain of poly(2-vinylpyridine) make them

alluring candidates for applications requiring chelation of heavy metals. In this study, we

report the use of poly (2-vinylpyridine) (P2VP) homopolymers of varying molar masses

for the stabilization of gold nanoparticles for the first time. A study based on AFM, DLS

and UV-visible spectroscopy was conducted to establish a correlation of the molar mass

of P2VP with the size and distribution of the gold nanoparticles. Systematic and gradual

change in the absorbance intensity and shift in SPR band of gold nanoparticles were also

observed upon variations in treatment temperature, concentration of polymer, residence

time, pH, and electrolyte concentration. The results obtained by UV-visible spectroscopy,

AFM and DLS are complementary. The size of the P2VP-stabilized AuNPs was found to

be in the range of 20-130 nms. At last, the effect of the size of P2VP-stabilized AuNPs

(directly related to the molar mass of P2VP) on the drug-loading efficiency is evaluated.

2 Introduction

Incorporating nanoparticles (NPs) into polymeric matrices is a practical pathway to

engineer advanced functional materials with improved optoelectronic and physico-

chemical properties. Polymer matrices control the spatial arrangement of entrapped

nanoparticles, consequently, well-defined and more stable structures at micro and

nanoscale are obtained (Balazs et al., 2006; Bockstaller et al., 2005; Fahmi, Pietsch,

Mendoza, & Cheval, 2009; Galatsis et al., 2010; Grzelczak, Vermant, Furst, & Liz-

Marzán, 2010; Haryono & Binder, 2006; Huh, Ginzburg, & Balazs, 2000; Kao,

Thorkelsson, Bai, Rancatore, & Xu, 2013; H.-C. Kim et al., 2009; Sarkar &

Alexandridis, 2015; Shenhar, Norsten, & Rotello, 2005; Vaia & Maguire, 2007; Zhang,

Liu, Yao, & Yang, 2012) Study of significantly different properties of nano-sized

particles compared to their bulk materials is a captivating topic of the scientific

research (Aurélien et al., 2014; Carotenuto et al., 2000; Y. Mei, Lu, Polzer, Ballauff, &

Drechsler, 2007; Weibel, Caseri, Suter, Kiess, & Wehrli, 1991). Physical properties and

applications of NPs strongly depend on the particle shape, size, inter-particle distance,

38

and nature of the protecting group. In addition, the exploitation of size-dependent

properties of NPs open new prospects towards the development of novel functional

materials, such as photonic devices (i.e. single-electron transistors, supercapacitors, and

data storage devices) (Bockstaller & Thomas, 2003; Cheng et al., 2001; H.-C. Kim et

al., 2009), high performance catalysis (Henry, 2000), and effective chemical or

biochemical sensors (Bruns & Tiller, 2005). These properties can be manipulated by

use of polymers and surfactants, through the immobilization and the assembly of the

nanoparticles in a suitable medium. Furthermore, nanoparticles of uniform size are

required for their better efficiency (Carotenuto, 2001; Carotenuto et al., 2000; Crooks et

al., 2001; B. J. Kim et al., 2006; B. J. Kim, Fredrickson, & Kramer, 2008; Lekesiz et

al., 2015; Luo, Zhang, Zeng, Zeng, & Wang, 2005; Y. Mei et al., 2007; Odegard,

Clancy, & Gates, 2005; Shan & Tenhu, 2007; Youk et al., 2002).

A number of organic materials such as low molecular weight (LMW) surfactants and

polymers have been used as protective agents for the preparation of AuNPs. Polymers

offer several advantages compared to LMW surfactants with respect to stability of the

self-organized structures, since the complexation of the polymer ligands with metal

particles is crucial before reduction and small amount of polymer reduces the particle size

dramatically (Abraham et al., 2007; Daniel & Astruc, 2004; Gandubert & Lennox, 2005b;

Perrault & Chan, 2009; Schaaff & Whetten, 2000; Shan & Tenhu, 2007; Toshima &

Yonezawa, 1998; Youk et al., 2002).

Polymer matrices offer control over particle size and also protect the surface of the

nanoparticles against aggregation. Controlled sizing and stability of polymer coated NPs

inspired several studies dedicated to novel synthetic routes for linking polymers to metal

nanoparticles, and the investigations of enhancement of properties and potential

applications of these hybrid materials (Bockstaller et al., 2005; Jiang et al., 2009b; H.-C.

Kim et al., 2009; Toshima & Yonezawa, 1998). Recently, metal nanoparticles, especially

gold nanoparticles (AuNPs), have been investigated extensively due to their unique

electronic, optical, catalytic properties and as well as new delivery vehicles for

biomedical applications. AuNPs have been used extensively for drug delivery, bio

imaging, diagnostic and other therapeutic applications because of their easy synthesis,

39

higher biocompatibility, lower toxicity and opportunity for surface modification (Gindy,

Panagiotopoulos, & Prud'homme, 2008; Pooja et al., 2015; Shukla et al., 2005).

Nanoparticles stabilized with polymer are particularly important for drug delivery owing

to their increased drug loading efficiency, biological stability, and extended in vivo

circulation (Kwon & Kataoka, 2012). Polyaspartic acid has been used as a reducing and

functionalizing agent for the synthesis of doxorubicin loaded gold nanoparticles

(Khandekar, Kulkarni, & Devarajan, 2014). Another research group used chitosan as

reducing/ stabilizing agent for stabilization of gold nanoparticles for insulin loading and

delivery (Bhumkar, Joshi, Sastry, & Pokharkar, 2007).

Polymers containing pyridine moiety have been used as a protective agent in the form of

ligands (Carotenuto et al., 2000; Lekesiz et al., 2015; Shan & Tenhu, 2007; Walker et al.,

2001). Poly(2-vinylpyridine) (P2VP) is an excellent candidate for coordination

chemistry. Nitrogen atoms of the pyridine moiety have strong affinity for the metal ions

and metallic nanoparticles, thus, restrains the aggregation of the metal nanoparticles

through steric stabilization (Jang et al., 2012; Lekesiz et al., 2015; Mössmer et al., 2000;

Voulgaris et al., 1999; Youk et al., 2002). Protective agent such as P2VP envelopes the

nanoparticles and any direct connection of metal particles to each other is impeded. In

conjunction with steric stabilization, P2VP prompts the reaction at ambient temperature

that results in reduction of particle size. A random coil conformation of P2VP in solution

may take part in some type of association with the metal atoms, therefore increasing the

probability of nucleus formation. Moreover, in the presence of P2VP average particles

size decreases along with the rate of spontaneous nucleation that results in higher number

of nuclei during the nucleation burst, consequently, total number of nanoparticles

increase (Carotenuto et al., 2000; Gandubert & Lennox, 2005b; Youk et al., 2002). There

are several studies of stabilization of NPs with block copolymer containing P2VP

(Bronstein et al., 1999; Gandubert & Lennox, 2005b; Jang et al., 2012; Ribbe, Okumura,

Matsushige, & Hashimoto, 2001; Yu et al., 2008). Up to best of our knowledge, P2VP

homopolymers have never been employed for stabilization of NPs.

In this study, we report on stabilization of gold nanoparticles by P2VP homopolymers of

varying molar masses. The effect of molar mass on the size and distribution of gold

40

nanoparticles is investigated systematically. Gold nanoparticles are prepared by reduction

of tetrachloroauric acid trihydrate (HAuCl4.3H2O) with sodium borohydride at ambient

temperature in the presence of P2VP. Analyses of the prepared P2VP-stabilized AuNPs

were conducted by UV-Visible spectroscopy, AFM and DLS. Effect of different external

parameters such as residence time, concentration of polymers, temperature, pH and

concentration of salt on the size and stabilization of P2VP-stabilized AuNPs is evaluated.

At last, the effect of size of the AuNPs (directly related to molar mass of P2VP) on the

drug-loading efficiency is appraised.

2.1 Experimental

2.1.1 Materials and Instrumentation

Poly(2-vinylpyridine) (P2VP) of various molar masses were purchased from polymer

standards services (Mainz, Germany) (Table 2.1). Tetrachloroauric (III) acid trihydrate

(HAuCl4.3H2O) > 99.9 % (Sigma Aldrich, USA) was the starting material for the

synthesis of gold nanoparticles. NaBH4 >95.0 % (TCI, Tokyo, Japan) was used as

reducing agent for HAuCl4.3H2O and HPLC grade methanol >99.9 % (RCI Labscan

Limited, Thailand) was used as solvent. All the reagents were used without further

purification.

A digital pH meter model 510 (Oakton, Eutech, USA) equipped with a reference

Ag/AgCl electrode and a glass working electrode was used.

UV–visible spectra were recorded with a Shimadzu UV-1800 series spectrophotometer

(Kyoto, Japan) equipped with a double beam compartment operated at 1 cm path length

quartz cuvette. The wavelength range was from 190 nm to 800 nm.

FTIR spectra were recorded on a FTIR Bruker Vector 22 spectrometer (Germany) using

KBr pellet method. All the analyses were performed in the mid IR range (400-4000 cm-1

).

Ten scans were used to obtain the spectral resolution of 0.1 cm–1

.

Comparative corresponding radius distribution of P2VP-stabilized AuNPs were studied

by a dynamic light scattering (DLS) Laser Spectroscatter-201 system (RiNA GmbH

41

Berlin, Germany), equipped with He-Ne laser, 690 nm light source and an output power

in the range of 10–50 mW. The CONTIN algorithm was used to analyse autocorrelation

functions in order to obtain hydrodynamic radius (RH). All experiments were performed

at 25 °C with an auto-piloted run of 50 measurements (20 s for single measurement) with

a wait time of 1 sec.

The topographical images of the P2VP-stabilized gold nanoparticles were recorded by

atomic force microscopy (AFM), Agilent 5500 (Arizona, USA). Triangular soft silicon

nitride cantilever (Veeco, model MLCT-AUHW) with a nominal value of 0.01 Nm-1

and

0.1 Nm-1

for the spring constant was used in the tapping mode for all measurements.

Samples were prepared by putting a drop of freshly prepared solution on the surface of

silicon wafer, and subsequently dried in air.

Table 2.1: Molecular weight and polydispersity index of P2VP homopolymers, as

provided by manufacturer

Sample Mn

(g/mol)

Mw

(g/mol) Mp (g/mol)

PDI

Mw/ Mn

P2VP1K 668 901 839 1.35

P2VP2K 1640 1910 1820 1.16

P2VP5K 4880 5460 5080 1.12

P2VP10K 10700 11100 11000 1.04

P2VP20K 22000 23500 22000 1.07

2.1.2 Preparation of P2VP Coated Gold Nanoparticles

Synthesis of P2VP-coated AuNPs was accomplished by using a two-phase system

consisting of methanol and water. The concentrations of the solution of P2VP,

HAuCl4.3H2O and NaBH4 were 0.1, 0.25 and 16 mM respectively. Solutions were mixed

in a volume ratio of 1:20:0.1. Aliquot of P2VP solution was added into a stirred aqueous

solution of HAuCl4.3H2O. Few drops of aqueous solution of NaBH4 were added into the

42

reaction mixture after 15 min and solution was continuously stirred for 48 h. Solutions of

purple and ruby red color indicated the formation of gold nanoparticles.

The pH of P2VP-stabilized gold nanoparticles is adjusted by dilute solutions of HCl and

NaOH.

Solutions of NaCl of varying concentration are mixed with P2VP-stabilized AuNPs in a

ratio of 1: 1.

Naringin, a natural flavonoid, was selected as model drug for loading in the

nanoparticles. Naringin was dissolved in methanol. Four millilitres of P2VP-stabilized

AuNPs are mixed with 2 mg of Naringin in order to have concentration of 0.5 mg/mL of

drug. Samples were stirred for 24 h at ambient temperature. Samples were then

centrifuged at 10000 RPM for 25 minutes. Supernatants were collected and analyzed for

free Naringin. UV-visible spectrophotometer was used for quantification of Naringin.

Naringin solutions of varying concentration (0.00391-0.0625 mg/mL) were used for

construction of calibration curve and UV-visible spectra were recorded at 284 nm. Blank

does not show any absorbance at 284 nm.

2.2 Results and Discussion

Poly (2-vinyl pyridine) (P2VP) has been used for the fabrication of novel functional

materials because of its ability to coordinate with metal nanoparticles (Jang et al., 2012;

Lekesiz et al., 2015; Mössmer et al., 2000). Coordination ability of P2VP arises from the

lone pair of electron at the nitrogen of pyridine moiety. Capability of coordination with

the metal nanoparticles increases with length of the polymer chain, thus, their reducing

ability. Therefore, the molar mass of P2VP plays a vital role in the formation, sizing and

stability of gold nanoparticles.

P2VP-stabilized AuNPs were directly subjected to UV-visible spectral analysis. A

systematic change in the colour of the solutions from purple to red indicates that the size

of AuNPs decreases with increase in molar mass of P2VP (Figure 2-1A). Figure 2-1B

depicts the successful preparation of P2VP-stabilized AuNPs by using a wide range of

43

molar mass of P2VP homopolymers. Formation of AuNPs is indicated by surface

plasmon resonance (SPR) band between 500-550 nm (David I. Gittins & Frank Caruso,

2001). Systematic and gradual change in the absorbance intensity and blue shift in SPR

band of P2VP-stabilized AuNPs with increase of molar mass indicates the reduction in

size of the NPs. Weak absorption of NPs stabilized with lower molar mass P2VPs at 550

nm indicates incomplete reduction. Understandably, reducing ability of P2VP increases

with molar mass due to availability of more nitrogen on polymer chain.

Figure 2-1: Effect of molar mass of P2VP on size and stability of P2VP-stabilized

AuNPs; A) Colour of solution and size; B) UV-vis spectra

44

To confirm the formation of P2VP-stablized AuNPs, FTIR spectroscopy is employed.

Figure 2-2 illustrates a comparison of the FTIR spectra collected for AuNPs stabilized by

P2VP of varying molar masses. The stretching absorption band at 1589 cm-1

corresponds

to C-N bond of pyridine ring. Stabilization of AuNPs by P2VP is confirmed by

disappearance of peak at 1589 cm-1

. Shifting of peak to 1650 cm-1

might be the result of

interaction of gold with the basic nitrogen centers in the polymer backbone.

Figure 2-2: FTIR spectra of unstabilized AuNPs (> 10,000 nm), P2VP (5000 g/mol) and

P2VP-stabilized AuNPs

Furthermore, the size and morphology of P2VP-stabilized AuNPs is determined by

atomic force microscopy (AFM) and dynamic light scattering (DLS). Spherical shaped

P2VP-stabilized nanoparticles are widely separated for all the samples, Figure 2-3 A-E.

As can be noticed, size and polydispersity of P2VP-stabilized AuNPs decreased with

increase of the molar mass of P2VP. Dependence of the size of P2VP-stabilized AuNPs

45

on molar mass of P2VP is further confirmed by DLS analysis. Hydrodynamic radii of

P2VP-stabilized AuNPs decreased as the molar mass of stabilizing P2VP increased.

Hydrodynamic radii of P2VP-stabilized AuNPs as obtained by DLS are 58, 54, 17, 15,

and 11 nms for molar masses of 1, 2, 5, 10, and 20 kg/mol, respectively, Figure 2-4. It is

pertinent to mention here that the size of the nanoparticles should correspond to the

double value of the hydrodynamic radii as obtained by DLS. Sizes of P2VP-stabilized

AuNPs are compared as a function of the molar mass of P2VP. The vital role of molar

mass of stabilizing P2VP in the sizing and polydispersity of AuNPs is established by

complementary results of UV-vis, FTIR, DLS, and AFM.

Figure 2-3: AFM images of P2VP-stabilized AuNPs, showing the average particle sizes;

A) AuNPs/P2VP1K; 125 nm, B) AuNPs/P2VP2K; 96 nm, C) AuNPs/P2VP5K; 43 nm, D)

AuNPs/P2VP10K; 32 nm, E) AuNPs/P2VP20K; 28 nm. The scale bar represents 0.25 µm

on all images

46

Figure 2-4: Physical characterization of P2VP stabilized AuNPs by DLS. (A) Dynamic

light scattering results of P2VP2K illustrating the experimental conditions i.e., the mean

autocorrelation function, monodispersity and radius plot (I to III), respectively. (B)

Comparative corresponding radius distribution of P2VP-stabilized AuNPs, effect of

molar mass on the size distribution. All experiments were performed with an auto–piloted

run of 50 measurements (20 s for single measurement) with a wait time of 1 s at 25 °C.

In order to evaluate long term stability of P2VP-stabilized AuNPs, samples were kept at

ambient temperature for several months and are monitored by UV-vis spectroscopy from

time to time. It is observed that the P2VP-stabilized-AuNPs are stable till 6 months for all

the molar mass range of P2VP used in this study. Enhanced intensity of SPR band with

residence time indicates the improved stability of AuNPs. As a typical example, stability

of P2VP10K-AuNPs measured from time to time for six months is shown in Figure 2-5.

47

Figure 2-5: Stability of the P2VP-stabilized AuNPs as a function of residence time as

indicated by UV-vis spectroscopy

AuNPs were synthesized by variations in the volume (0.025-1.0 mL) of 0.1 mM P2VP

solution to assess the effect of number of nitrogen in the polymer chain on the particle

size. The effect of concentration of P2VP on the sizing and stability of AuNPs is

demonstrated by taking an example of P2VP5K-stabilized AuNPs. Figure 2-6A

demonstrates the change in the color of solution from purple to red on addition of

different volumes of P2VP solution. As can be noticed that 0.05 mL of 0.1 mM of P2VP

solution is the minimum amount required for formation of AuNPs. The color of the

solutions gave an indication of the different sizes of the P2VP-stabilized AuNPs. Broad

peak of the UV spectrum of AuNP stabilized with 0.05 mL of 0.1 mM P2VP solution

indicates the polydispersity of the particles in solution, Figure 2-6B. Increase in the

concentration of P2VP leads to narrow peaks, which is an indication of monodispersity of

the nanoparticles in solution. Hydrodynamic radii as obtained by DLS analysis also

endorse the results of UV-vis spectroscopy. Smaller amount of P2VP in the solution was

not enough to fully reduce all the gold in the solution and resulted in trimodal

48

distribution. Control over the size distribution of the AuNPs increased with increase in

the amount of P2VP, Figure 2-6C. Hence, certain amount of polymer is required to fully

stabilize the nanoparticles that depend upon the average number of nitrogen present in the

polymer chain. The ability of P2VP to stabilize AuNPs increases with number of nitrogen

available for reduction of gold. Better control over sizing of P2VP-stabilized AuNPs with

increase in the molar mass (Figure 2-4) supports the outcome of concentration profile of

P2VP on the stability and sizing of AuNPs.

49

Figure 2-6: Effect of the concentration of P2VP5K on the stability, size and distribution of

AuNPs; A) Visual difference in colour, B) UV-Vis spectroscopy, C) Dynamic light

scattering

Stability of P2VP-stabilized AuNPs as a function of temperature is monitored by keeping

the samples at desired temperature for 10 minutes. Samples were brought to ambient

temperature without any external cooling/heating. Thereafter, UV-vis spectra of the

samples were recorded. Increased intensity of the absorption maxima, Amax of SPR band

by increasing treatment temperature is indication of enhanced relative stability of AuNPs.

Increased stability with elevated temperature might be due to better conversion of gold

ions into gold nanoparticles. As a typical example, the effect of temperature treatment on

the sample stabilized with P2VP2K is shown in Figure 2-7.

50

Figure 2-7: The effect of temperature on the stability of P2VP2K-stabilized AuNPs as

shown by UV-vis spectroscopy

Effect of pH on the stability of P2VP coated gold nanoparticles was monitored via

changes in the SPR band in UV-visible region. Figure 2-8 illustrates absorption spectra of

aqueous solution of P2VP-AuNPs as a function of pH values. The position and shape of

the surface plasmon band did not change with variations in pH from 2 to 12, confirming

that flocculation and aggregation have been protected over a wide range of pH. A small

decrease in absorption maxima for acidic region (pH < 7) indicated that the sphericity of

AuNPs is not conserved and agglomeration occurred. Persistence of single plasmon peak

is a sign of the stability of AuNPs as a function of pH. Aggregation of AuNPs is

augmented by the adsorption of [AuCl4]- over the surface of AuNPs. Thus, the

stabilization of AuNPs with increase in pH can be explained in context of [AuCl4]-

forming lesser reactive [AuCl(4-n)(OH)n]- complex with OH

-, where (n) increases with

the pH (Badawy et al., 2010a; Gandubert & Lennox, 2005c).

51

Figure 2-8: Effect of pH on P2VP stabilized gold nanoparticles as shown by UV-vis

spectroscopy

It is generally known that addition of electrolyte can result in agglomeration of the

nanoparticles into large aggregates. A detailed study was carried out on the effect of the

concentration of electrolytes on the stability of P2VP-coated gold nanoparticles. Samples

were monitored by UV-visible spectroscopy. Equal volumes of P2VP-AuNPs solution

and various concentrations of NaCl aqueous solutions were mixed. Surprisingly, two

different trends were observed on the addition of solutions of different concentrations of

electrolytes. P2VP-stabilized AuNPs based on higher molar masses (5, 10 and 20 kg/mol)

have no appreciable change in the intensity of absorption maxima at various electrolyte

concentrations, indicating that longer polymer chains cover the AuNPs surface

completely, hampering the effect of ions of salt on AuNPs. Cations in solution may attach

to the free nitrogen atoms in the polymer chain that might be the reason of different

origin of absorption peaks, Figure 2-9 B (Badawy et al., 2010a; Lévy et al., 2004; Yusa et

al., 2007).

52

AuNPs stabilized with P2VP of lower molar mass (1 and 2 kg/mol) behave differently by

addition of NaCl solutions of different concentrations, Figure 2-9 A. Enhanced stability

of P2VP-stabilized AuNPs by addition of dilute solutions is indicated by increase in the

absorption maximum. This may be due to high steric stabilization caused by smaller

chain lengths of P2VP. In polymer coated nanoparticles, steric repulsive forces and

electrostatic repulsive forces exist simultaneously. Combination of steric and electrostatic

forces allowed for the stabilization of nanoparticle dispersions over a wide size range, by

means of polymer coating containing one or more ionic charges. The electrostatic

repulsive forces are dominant over the attractive forces, and thus suppressing

agglomeration under such conditions leading to stable dispersions. However, for low

molar mass samples intensity of absorption maxima of P2VP-stabilized AuNPs decreased

as the concentration of electrolyte solution increased beyond 0.1 M. This means that the

energy barrier to prevent agglomeration decreased with increasing ionic strength of

solution (Gandubert & Lennox, 2005c; Jiang, Oberdörster, & Biswas, 2009a).

53

Figure 2-9: Effect of various salt concentrations on P2VP coated gold nanoparticles as

shown by UV-vis spectroscopy, A) P2VP10K-Au NPs; B) P2VP2K-Au NPs

54

To evaluate the effect of molar mass of P2VP on drug loading efficiency of AuNPs, a

natural flavonoid Naringin was used as a model drug. Calibration curve of Naringin was

found to be linear in a concentration range of 0.00391-0.0625 mg/mL for absorbance at

284 nm, Figure 2-10 A. The drug-loading efficiency increases with decrease in the size of

NPs that is directly related to the molar mass of P2VP. Highest molar mass P2VP used in

this study (20K) is found to be the best with regard to drug-loading efficiency. Drug-

loading efficiency of P2VP-stabilized AuNPs decreased with the decrease in the molar

mass of P2VP, Figure 2-10B.

55

Figure 2-10: A) Calibration curves for quantification of Naringin in concentration range

of 0.00391-0.0625 mg/mL; B) % drug-loading efficiency of P2VP-stabilized AuNPs

2.3 Conclusion

Molar mass of P2VP has enormous effect on stabilization and size of AuNPs. Reducing

ability of P2VP increased with increase of its molar mass due to availability of more

nitrogens, that results in smaller sized AuNPs. The results obtained by UV-vis, FTIR,

AFM, and DLS complement each other. Furthermore, effect of variations in

concentration of P2VP, residence time of stabilized AuNPs, temperature treatment,

addition of electrolyte, and pH are evaluated. P2VP-stabilized AuNPs remained intact up

to six months. The stability of P2VP-stabilized AuNPs increased after treatment at

elevated temperatures and at higher pH values. Different trends were found for

stabilization of AuNPs on addition of electrolytes that seem to be dependent upon molar

mass of P2VP. Furthermore, it has been shown that the drug-loading efficiency of P2VP-

stabilized AuNPs increases with decrease in the size of the nanoparticles, which is

directly related to the molar mass of stabilizing P2VP.

56

Chapter 3

Polystyrene-block-poly(2-vinylpyridine)-conjugated

silver nanoparticles as colorimetric sensor for

quantitative determination of Cartap in aqueous media

and blood plasma

57

Abstract

Development of novel materials for different analytical applications such as optical

sensors is one of the major topics of modern scientific research. In this study, a

nanosensor based on highly stable silver nanoparticles (AgNPs) conjugated with

polystyrene-block-poly(2-vinyl pyridine) [PS-b-P2VP or P(S-VP)] copolymer was

synthesized using two-phase one pot protocol. The nanosensor was characterized by UV-

visible spectroscopy, zetasizer, FTIR and AFM. Polystyrene-block-poly(2-vinylpyridine)-

conjugated silver nanoparticles [P(S-VP)-AgNPs] were further utilized as colorimetric

sensor for thiocarbamate pesticide, cartap. P(S-VP)-AgNPs nanosensor allowed for rapid

and quantitative detection of cartap in concentration range of 0.036-0.36 μgL−1

with

detection limit as low as 0.06 μgL−1

. The prepared sensor efficiently detected cartap in

presence of other interfering pesticides. P(S-VP)-AgNPs demonstrated great potential for

in situ detection of cartap in water and blood plasma.

3 Introduction

Use of pesticides in agriculture and other related fields is increasing significantly.

However, besides their beneficial effects for rapid and safe growth of crops, they can

persist in the environment to cause pollution. Presence of pesticides beyond their

acceptable limits in surface water is noticed in different cities of the world. Agricultural

run-off and vector control sprays are the major sources of unacceptable concentration of

pesticides in fresh water. These pesticides not only influence the aquatic life but also

have adverse effects to human beings. After intended application, many recalcitrant and

non-biodegradable pesticides survive in the environment for prolonged period of time

(Casida & Quistad, 1998; Y. Kim, Jung, Oh, & Choi, 2008). Water soluble pesticides

such as carbamates and thiocarbamtes are extensively used for safer and rapid growth of

agricultural and ornamental plants. Hence, development of simple, effective and

inexpensive analysis procedure is imperative for specific and quantitative detection of

pesticides in water. A number of techniques such as chromatography (Park et al., 2015),

electroanalysis (Everett & Rechnitz, 1998), photoluminescence (Yuan, Ma, & Xu, 2016)

58

and fluorescence (Ahmed, Khalid, Shah, & Shah, 2016; Cao, Zhang, Ma, Liu, & Yang,

2013; Guo et al., 2014) have been employed for this purpose till date; however, these

techniques require expensive instrumentation, long analysis time, complicated procedures

and trained technicians. Above-mentioned factors are highly unwanted for rapid and

routine screening procedures.

Remarkable optical properties of metallic NPs due to collective oscillation of surface

electrons in a conduction band after interaction with electromagnetic radiation (EMR)

make them tremendous candidate for their application as nanosensors (Aragay, Pino, &

Merkoçi, 2012; Ateeq et al., 2015). The optical response of NPs depends upon size,

shape and interparticle distances. Particularly, silver and gold have gained extensive

consideration in recent years. These NPs show surface plasmon resonance (SPR) band in

visible region (380–750 nm). During the recognition process, the surrounding

environment of NPs changes due to analyte interaction with different groups on the

surface of NPs that causes a shift in surface plasmon band.

Cartap, S,S-[2-(dimethylamino)-1,3-propanediyl] dicarbamothioate, a thiocarbamate, is a

precursor or analogue of natural insecticide nereistoxin that directly attacks nervous

system of insects, included in group 14 of IRAC MoA classification (Sparks & Nauen,

2015). Cartap was considered non-toxic earlier, however, recent cases of cartap poisoning

have prompted for development of specific method for its detection and quantification.

Effects of its access on humans include headache, palpitation, flushed face, irritation of

nose, throat, eyes and skin (Eldefrawi, Bakry, Eldefrawi, Tsai, & Albuquerque, 1980;

Kurisaki et al., 2010; Liao et al., 2003; Nagawa, Saji, Chiba, & Yui, 1971; Raymond-

Delpech, Matsuda, Sattelle, Rauh, & Sattelle, 2005; Vivek, Veeraiah, Padmavathi, Rao,

& Bramhachari, 2016). It has been shown that ocular exposure of cartap following

respiratory failure, mainly due to calcium-mediated diaphragmatic contracture rather than

neuromuscular blockage in rabbits (Kumar, Amalnath, & Dutta, 2011; Liao et al., 2006).

Basically, cartap induces generation of reactive oxygen species through a calcium

dependent mechanism that may be responsible for contracture and myofiber injury of

diaphragm, ultimately leading to respiratory failure and death (Harsha, Abhilash, &

Hansdak, 2013; Kurisaki et al., 2010).

59

In chemosensing, secondary interactions play a vital role such as hydrogen bonding (Boal

et al., 2000), van der waals forces (Patil et al., 1997), π-π stacking (J. Jin et al., 2001),

host-guest mechanism (J. Liu et al., 1999), charge transfer (Naka et al., 2003),

electrostatic attraction (Caruso et al., 1998) and antigen-antibody interactions (Shenton,

Davis, & Mann, 1999) etc. Silver nanoparticles stabilized through nitrogen containing

compounds have been used for the detection of anions. Positive charges are created on

the surface of the compound due to donation of electron pairs by nitrogen. Various

natural macromolecules like proteins, flavonoids, liposomes and polysaccharides as well

as synthetic macromolecules such as polymers have been employed for construction of

nanosensors (Fang et al., 2011; Gandubert & Lennox, 2005a). In this study, polystyrene-

block-poly(2-vinylpyridine), P(S-VP), copolymer has been used for stabilization of silver

nanoparticles (AgNPs). Pyridine moieties of P2VP block of copolymer have basic

nitrogens that have tendency to chelate metal nanoparticles, hence, prevent aggregation

and promote stabilization. P2VP has a very low contact angle with Au (9°), and 20 nm

AuNPs stabilized at a PS-P2VP interface tend to diffuse into P2VP (Kunz et al., 1993).

Successful utilization of P2VP homopolymers for stabilization of AuNPs is shown in our

recent publication (Rahim et al., 2017). Precise control of the particle location within

P2VP domain of P(S-VP) block copolymer stabilized AuNPs has been demonstrated

(Chiu et al., 2005). The stabilization of AuNPs by 4-(dimethylamino)pyridine is also

reported (Gandubert & Lennox, 2005a).

Herein, we report a new and facile one-pot robust approach for synthesis of thermally

stable P(S-VP)-conjugated silver nanoparticles (AgNPs). P(S-VP)-AgNPs are

synthesized by reduction of silver nitrate in presence of sodium borohydride with P(S-

VP). Specifically, we demonstrate the utilization of chelating ability of pyridine moiety

of polymer for stabilization of AgNPs. The synthesized AgNPs were characterized by

UV-visible spectroscopy, zetasizer, FTIR and AFM. Further, the P(S-VP)-AgNPs were

utilized for specific monitoring and quantification of cartap in presence of other

interfering species and in real samples such as ground water and blood plasma. The

proposed procedure offers fast, economical and sensitive method for detection of cartap

in water and physiological fluids for routine analysis.

60

3.1 Experimental


Polystyrene-block-poly(2-vinyl pyridine) PS26K-b-P2VP4.8K (PDI = 1.15) was purchased

from polymer source inc (Quebec, Canada). Silver nitrate (AgNO3) (Sigma Aldrich,

USA) was the starting material for the synthesis of silver nanoparticles. NaBH4 (TCI,

Tokyo, Japan) was used as reducing agent for AgNO3. HPLC grade methanol (MeOH)

and toluene (RCI Labscan limited, Thailand) were used as solvents. All the reagents were

used as received. Pesticides samples were collected from Industrial Analytical Centre

(IAC), International Centre for Chemical and Biological Sciences (ICCBS), University of

Karachi, Pakistan.

Glassware were washed with aqua regia, oven-dried and rinsed with deionized water and

methanol prior to use.

A digital pH meter (Oakton, Eutech) model 510, with a Ag/AgCl reference electrode and

a glass working electrode was used to adjust pH of P(S-VP)-AgNPs solutions.

UV–visible spectra were recorded with a double beam Shimadzu UV-1800 series

spectrophotometer operated at a wavelength range of 190-800 nm using quartz cuvette of

one centimeter path length.

Particle size distribution and zeta potential of P(S-VP)-AgNPs before and after treatment

with cartap were determined by zetasizer, Nano-ZSP (Malvern Instruments). The analysis

was performed at a scattering angle of 90° at a temperature of 25 °C using disposable

cuvette for zetasizer and dip cell cuvette for zeta potential studies.

FTIR spectra were recorded on a Bruker Vector 22 spectrometer in the mid IR range

(400-4000 cm-1

) using KBr pellet. Ten scans were used to attain the spectral resolution of

0.1 cm–1

.

P(S-VP)-AgNPs topographical images were recorded by Agilent 5500 atomic force

microscope (AFM), (Arizona, USA). Triangular soft silicon nitride cantilever (Veeco,

61

model MLCT-AUHW) with a nominal value of 0.01 Nm-1

and a spring constant value of

0.1 Nm-1

in the tapping mode was used for all measurements. A drop of freshly prepared

sample was taken on the surface of silicon wafer, and subsequently dried in air.

3.1.2 Preparation of P2VP Coated Gold Nanoparticles

The synthesis of P(S-VP)-AgNPs was accomplished by using a two-phase system

consisting of methanol and toluene. The concentrations of solution of P(S-VP), AgNO3

and NaBH4 were 0.1, 1.0 and 4.0 mM respectively. The solutions were mixed in a

volume ratio of 1:30:0.1. Aliquot of P(S-VP) solution was added into a stirred aqueous

solution of AgNO3. Few drops of aqueous solution of NaBH4 were added into the

reaction mixture after 15 min and stirred continuously for 30 min. Appearance of yellow

solution indicated the formation of silver nanoparticles (AgNPs) stabilized by P(S-VP).

3.1.3 Spiking in Tap Water and Surface Runoff Water

Tap water was collected from university of Karachi. Cartap solution of 0.1 mM was

prepared in a mixture of methanol and tap water (60:40) to minimize the precipitation of

polymer in water. The interaction after mixing of 1:1 v/v solutions of cartap and P(S-

VP)-AgNPs was evaluated by UV-vis analysis. Same procedure was followed for surface

runoff water collected from the lawns of university of Karachi.

3.1.4 Spiking in Human Blood Plasma

Blood sample was collected in heparinized tube from a healthy human volunteer after

ethical approval from ethics committee of the center via venous puncture followed by

centrifugation at 4000 revolutions per minute (rpm) for 5 min at room temperature to

separate out plasma. Two different stock solutions were prepared taking 1.0 mL of

plasma and 2.0 mL of P(S-VP)-AgNPs, diluted with methanol up to 5 milliliter. 1.0 mL

of plasma containing AgNPs stock solution was analyzed without adding cartap while the

other solution was spiked with 1 mL of 0.1 mM cartap solution.

62


3.2.1 Synthesis and characterization of P(S-VP)-AgNPs

Polystyrene-block-poly(2-vinylpyridine) (P(S-VP)-AgNPs) is an Amphiphilic block

copolymer that form spherical micelles with PS corona and P2VP core in toluene,

because toluene is a good solvent for PS while non-solvent for P2VP. On the other hand,

presence of nitrogen atoms in P2VP block makes it suitable for fabrication of metallic

nanoparticles in its domain.

Silver nanoparticles were obtained in two-phase system (toluene-methanol) by reacting

0.1 mM P(S-VP) and silver nitrate (AgNO3) in presence of sodium borohydride. P(S-

VP)-AgNPs were characterized by AFM, FTIR, zetasizer and UV–vis spectroscopy. The

colorless reaction mixture rapidly turns yellow that indicates reduction of silver ions and

formation of P(S-VP)-AgNPs. UV–visible spectra of the solution revealed an absorption

maximum at 428 nm (Figure 3-1). Typically, silver nanoparticles have an absorption

maximum between 400 and 450 nm (Solomon et al., 2007). The amount of conjugated

P(S-VP) was measured by centrifuging P(S-VP)-AgNPs solution at 14,000 rpm for 40

min. The supernatant was freeze-dried and the residue weighed. The results indicated that

conjugates contained about 80% by weight of initial amount of P(S-VP).

63

Figure 3-1: UV-visible spectrum of P(S-VP)-conjugated AgNPs

The P(S-VP)-AgNPs remained stable after incubation at 64 °C (boiling point of

methanol) for 10 minutes. The sample was brought down to ambient temperature without

external cooling. Thereafter, a UV-vis spectrum of the sample was measured.

Enhancement in absorption maxima (Amax) of SPR band of temperature treated sample is

the indication of relatively higher stability of AgNPs, might be due to better conversion

of silver ions into silver nanoparticles or increased solubility of high molar mass polymer

(Figure 3-2). The P(S-VP)-AgNPs were found to be stable for several months at ambient

temperature.

64

Figure 3-2: UV-visible spectrum of P(S-VP)-conjugated AgNPs after incubation of P(S-

VP)-conjugated AgNPs at 64 °C for 10 minutes (B.P. of methanol)

Generally, addition of electrolytes in NPs solution results in agglomeration of NPs (Bae,

Nam, & Park, 2002). A detailed study was carried out to elucidate the effect of the

concentration of electrolytes (0.01mM – 5M NaCl) on P(S-VP)-AgNPs stability,

monitored by UV-visible spectroscopy. P(S-VP)-AgNPs were stable over a wide range of

concentration of sodium chloride (0.01 mM to 1.0 mM). Aggregation was only observed

by addition of sodium chloride solution having concentration beyond 5.0 mM, attributed

to the aggregation effect by Cl-1

ions present in solution, Figure 3-3.

65

Figure 3-3: Electrolyte effect on P(S-VP)-conjugated AgNPs with various salt

concentration (0.01mM-5M)

3.2.2 P(S-VP)-AgNPs and cartap response

As mentioned earlier, P(S-VP) block copolymers tend to make micelles in toluene that is

good solvent for PS while non-solvent for P2VP. PS block makes the corona while P2VP

tend to be away from toluene making core of the micelles. AgNPs tend to be inside the

core since they also do not prefer a non-polar environment. In the process, when cartap is

present in the solution, it tends to be attracted towards positive charges on the P2VP

block inside core of micelles because of its resonating structure (Figure 3-4).

66

Figure 3-4: Schematic representation of cartap recognition of P(S-VP)-AgNPs through

electrostatic interactions

The mean size and size distribution of P(S-VP)-AgNPs and P(S-VP)-AgNPs/ cartap

solution were analyzed using zetasizer. The size distribution profile of P(S-VP)-AgNPs

and P(S-VP)-AgNPs/ cartap showed a mean diameter of 104.2±0.68 and 89.68±0.57 nm

with PDI of 0.22 and 0.08, respectively (Figure 3-5 A, B).

Figure 3-5: The size distribution by intensity A) of P(S-VP)-AgNPs avg size: 104.2±0.68

nm, PDI: 0.22; B) P(S-VP)-AgNPs/ cartap. avg. size: 89.68±0.57 nm, PDI: 0.08

67

Interestingly, the mean diameter of P(S-VP)-AgNPs turned out to be more homogeneous

after addition of cartap, endorsed by AFM analyses too. P(S-VP)-AgNPs exhibited an

irregular assemblage shape (80-120 nm), Figure 3-6 A. While structure of P(S-VP)-

AgNPs/ cartap has a regular shape, and size apparently decreased (60-90 nm) as shown in

Figure 3-6 B. The regularity in the shape and reduction in size of the NPs by addition of

cartap can be attributed to the balancing of the surface charges.

Figure 3-6: Atomic force micrographs (AFMs) A) P(S-VP)-AgNPs (80-120 nm); B) P(S-

VP)-AgNPs/cartap (60-90 nm)

Zeta potential (surface charge) reveals the interactions of nanoparticle with analyte and

surroundings. It can greatly influence particle stability through electrostatic repulsion

between particles. The surfaces of P(S-VP)-AgNPs have a positive charge of 20.8 mV,

whereas P(S-VP)-AgNPs/ cartap exhibit zeta potential of 27.7 mV (Figure 3-7 A,B). It

seems that positive surface charges on P(S-VP)-AgNPs are neutralized by cartap through

electrostatic interactions (Figure 3-4).

68

Figure 3-7: Zeta potential distribution A) P(S-VP)-AgNPs; B) P(S-VP)-AgNPs/Cartap

FTIR studies of P(S-VP), P(S-VP)-AgNPs, and P(S-VP)-AgNPs/ cartap were performed

to have a deeper understanding of the mechanism of NP formation and recognition of

cartap in solution. A comparison between FTIR spectra of P(S-VP), P(S-VP)-AgNPs, and

P(S-VP)-AgNPs/cartap suggested that nitrogen atoms in the backbone of polymer

stabilized AgNPs, since C=N stretching vibration peak at 1592 cm-1

of P(S-VP)

disappeared upon the formation of AgNPs, while the rest of the characteristics peaks for

P(S-VP) are present (Figure 3-8). A new peak appeared at 1383 cm-1

that indicates the

formation of AgNPs. The FTIR spectra of cartap have C=O stretch at 1680, N-H stretch

3301 and 3242, N-CH3 stretch at 2979, and C-S stretching peaks were observed between

1187 to 1017 cm-1

. Interestingly, C=O, N-H, N-CH3 and C-S peaks of cartap were absent

in a mixture of P(S-VP)-AgNPs/ cartap, while the peak at 1383 cm-1

was still present and

a new peak at 1630 cm-1

appeared that indicates the formation of C=N bond. The

disappearance of the characteristic cartap peaks indicated the interaction of cartap with

polymer instead of silver.

69

Figure 3-8: FTIR spectra of P(S-VP), Cartap, P(S-VP)-AgNPs, and cartap treated

P(S-VP)-AgNPs

The size, surface charge, and morphology of P(S-VP)-AgNPs were greatly influenced by

adsorption of cartap. Silver ions adsorb onto the external surface of P(S-VP) and get

chelated with the nitrogen atoms of P2VP through complexation. The process changes

the surface electric charge of P(S-VP) because partial positive charges are created on the

surface by donation of the lone pair by nitrogen atom of P2VP. These positive charges on

P(S-VP)-AgNPs interact with negative charge of oxygen atoms of carbonyl group of

cartap molecules, indicated by the appearance of -C=N stretching peak at 1630 cm-1

in

FTIR spectra. The negative charges on cartap appear due to resonance as shown in

Scheme 3-1. Consequently, a decrease in size and surface charge of cartap treated P(S-

VP)-AgNPs is observed compared to P(S-VP)-AgNPs.

70

Scheme 3-1: Resonating structure of Cartap

3.2.3 Spectroscopic recognition of cartap

The recognition behavior of P(S-VP)-AgNPs was assessed for various pesticides by

mixing equal volumes of NPs solution with 0.1 mM pesticide solutions. UV–vis spectra

were recorded instantly after mixing. The effect of tested pesticides such as cartap,

deltamethrin, alpha-cypermethrin, carbofuran, chlorfenapyr, clodinafop propargyl,

lambda-cyhlalothrin, diuron, imida-cloprid and lufenron on absorption intensity was

evaluated (Figure 3-9). Structures of the competitive pesticides used in this study are

depicted in Scheme 3-2.

71

Scheme 3-2: Structure of cartap and other interfering pesticides

72

No interaction of pesticides was evident with P(S-VP)-AgNPs except cartap that showed a

blue shift, decrease in absorption maxima. Difference in absorption behavior of cartap and

other pesticides indicates interaction between P(S-VP)-AgNPs and cartap. As mentioned

earlier, the reason for selective detection of cartap compared to other competing pesticides

is the resonating structure of cartap that resulted in negative charges on the carbonyl

oxygen. Hence, UV-visible spectrophotometric results of pesticides screening suggested

that P(S-VP)-AgNPs nanosensor are very selective for detection of cartap.

Figure 3-9: UV-visible spectra of P(S-VP)-AgNPs complexed with various pesticides

The synthesized P(S-VP)-AgNPs were slightly acidic in nature having pH in range of 5-

6.The effect of pH on SPR band was studied by varying pH in the range of 2–12 (Figure

3-10). The synthesized P(S-VP)-AgNPs were found to be stable in a pH range of 7-12,

however, particle agglomeration was noticed below pH 7 that increased with further

decrease in the pH. On the basis of these results it can be concluded that the P(S-VP)-

AgNPs act as an excellent nanosensor for cartap in basic environment.

73

Figure 3-10: Effect of pH on accumulation of P(S-VP)-conjugated AgNPs with Cartap

As a next step, analytical performance of the nanosensor in terms of quantification is

evaluated and a calibration curve is constructed by plotting absorbance at 410 nm vs

concentration of cartap. The data strictly followed beer‘s law and have a good linear

correlation in the range from 0.036-0.36 μgL−1

with the regression constant (R2) equal to

0.9940. The approximate limit of detection (LOD) of cartap is found to be 0.06 μgL-1

,

Figure 3-11. Although, detection limit of the proposed method is more than highly

sensitive chromatographic techniques such as HPLC, however far lower than other

reported spectrometric and GC-MS methods.

74

Figure 3-11: A) UV-visible spactra by using various concentrations of cartap with P(S-

VP)-AgNPs; B) Calibration curve for amount of cartap at 410 nm

75

The binding stoichiometry between P(S-VP)-AgNPs and cartap was 1:1 as obtained by

Job's plot, Figure 3-12. The comparison of reported detection methods for cartap by

different techniques with current study is presented in Table.

Figure 3-12: Job‘s plot for binding ratio.

Practical demand of chemosensor for any application is its specificity for the analyte in

presence of other interferents. It is observed that addition of nine different interfering

pesticides in similar quantity does not have any pronounced effect on cartap recognition,

Figure 3-13.

To explore the utility and efficiency of optimized cartap recognition system, P(S-VP)-

AgNPs were employed for cartap recognition in spiked tap water, surface runoff water

and human blood plasma.

All samples were spiked with 0.1 mM concentration of cartap before analysis. Figure

3-14 A indicates that the distinctive cartap recognition signal is observed in P(S-VP)-

AgNPs with 0.1 mM cartap spiked tap water. Same comparison for surface runoff water

with similar results is presented in Figure 3-14 B. However, in case of blood plasma, only

a hypochromic shift with a slight blue shift of 4-5 nm band is observed (Figure 3-14 C).

76

The results suggest that the proposed cartap recognition system can be effectively utilized

for detection of cartap in water treatment and blood testing.

Figure 3-13: Effect of interfering pesticides on cartap detection by P(S-VP)-AgNPs, 1:

deltamethrin, 2: Alpha-cypermethrin, 3: carbofuran, 4: chlorfenapyr, 5: Lambda-

cyhlalothrin, 6: diuron, 7: imidacloprid, 8: lufenron, 9: clodinafop propa

77

Table 3.1: Comparison of reported cartap detection methods with current study

Methods/

Materials

Analytical

ranges LoD

Interfering species Sample Comments Ref.

HPLC 50–400 pmol 10

pmol

- Water

specific electrochemical

detector is required

(Fisher, Xie, &

Loring, 1993)

GC-MS 0.05–5.0

μgL−1

10

μg L−1

Cartap metabolites

Human

serum

Expensive

instrumentation

(Namera,

Watanabe,

Yashiki, Kojima,

& Urabe, 1999)

Flourescence/

CB[7]-PAL

0.009-2.4

μgmL-1

0.0029

μgmL-1

Thiram, daminozide, promethazine

hydrochloride, diphenhydramine

hydrochloride, chlorphenamine, maleate

Grain,

vegetable

Expensive

instrumentation , tedious

extraction procedure

(X. Jing, Du,

Wu, Wu, &

Chang, 2012)

Flourescence/

AuNPs- CdTe QDs

0.01-0.50

mgkg-1

8.24

mgkg-1

Methamidophos, imidacloprid, methomyl,

carbaryl, acetamiprid Chinese

cabbage

Expensive

instrumentation , tedious

pre-treatment procedure

(Guo et al.,

2014)

Photoluminescene/

Au@Ag

nanoparticles

0.222-0.709

mgkg-1

0.0062

mgkg-1

Omethoate, aldicarb, amitraz, dichlorovos,

methamidophos, imidacloprid, triazophos,

methomyl, carbaryl,

acetamiprid

-

Expensive instrument,

pre-treatment, time

consuming

(Yuan et al.,

2016)

Colorimetry/

Au NPs 50–250 μgkg

−1 40 μgkg

−1

Omethoate, aldicarb, amitraz,

dichlorovos, methamidophos, imidacloprid,

triazophos,

methomyl, carbaryl

Tea,

kiwifruit,

rice, cabbage

low cost, tedious pre-

treatment procedure

(W. Liu et al.,

2015)

Colorimetry/

Au NPs

0.05–0.6

mgkg-1

0.04

mgkg-1

Omethoate, aldicarb, amitraz, dichlorvos,

methamidophos, imidacloprid, triazophos,

methomyl, carbaryl, acetamiprid

Cabbage, tea low cost, tedious pre-

treatment procedure

(W. Liu et al.,

2012)

Colorimetry/

Ag NPs

0.036-0.36

μgL−1

0.06

μgL−1

Deltamethrin, Alpha-cypermethrin, carbofuran,

chlorfenapyr, Lambda-cyhlalothrin, diuron,

imidacloprid, lufenron, clodinafop propargyl. Water, blood

plasma

Easy synthesis compared

to AuNPs, low cost,

more sensitive, No pre-

treatment of sample

This study

78

79

Figure 3-14: Effect of cartap on absorbance intensity of P(S-VP)-AgNPs A) tap water; B)

surface runoff water; C) human blood plasma

3.3 Conclusion

In this study, novel P(S-VP)-AgNPs based nanosensor is reported via two phase one pot

protocol for the rapid quantitative assay of pesticide, cartap. P(S-VP)-AgNPs and its

interaction with cartap was studied using UV-visible spectroscopy, FTIR, zetasizer and

AFM. It is found that about 80% by weight of initial amount of P(S-VP) is used in

conjugation with AgNPs. P(S-VP)-AgNPs based nanosensor is found to be selective

towards cartap compared to other pesticides. It follows linear correlation with cartap

down to a concentration of 0.06 μgL−1

. Moreover, the detection system is consistent in

the presence of many interfering pesticides and ions in the real samples. The optimized

P(S-VP)-AgNPs based quantitative assay would potentially lead to more practical

applications because of its low cost, simple preparation, excellent selectivity, and low

detection limit.

80

Chapter 4

Enhancement in the electrochemical response of glassy

carbon electrode modified by poly(2-vinlypyridine)-b-

poly(methyl methacrylate) conjugated gold

nanoparticles for nicotine

81

Abstract

Investigation of the potential of poly(2-vinylpyridine-b-methylmethacrylate) coated gold

nanoparticles [P(2VP-MMA)-AuNPs] as an electrochemical sensor for nicotine is the

main focus of current study. P(2VP-MMA)-AuNPs were prepared and characterized by

UV-Vis, FTIR, AFM, and zetasizer. Further, P(2VP-MMA)-AuNPs were coated on a

glassy carbon electrode (GCE) for electrochemical detection of nicotine by cyclic

voltammetry. The effect of molar mass of individual P2VP block and total molar mass of

the block copolymer is evaluated in context of sensing ability of nicotine in both aqueous

and organic media. The electrochemical sensing of nicotine is significantly enhanced by

modification of GCE with P(2VP-MMA)-AuNPs.

4 Introduction

Nicotine, 3-(1-methyl-2-pyrrolidinyl) pyridine, is an alkaloid abundantly found in

nightshade family of plants. It is the major ingredient of tobacco and cigarettes. The

concentration of nicotine in tobacco is about 2 -8% (Selmar, Radwan, & Nowak, 2015).

Nicotine directly attacks the nervous system and hence is included in IRAC MoA group

4B (Sparks & Nauen, 2015). It is a potent para-sympathomimetic stimulant that acts as an

agonist at nicotinic acetylcholine receptors (nAChRs). The adverse effects of nicotine on

human health includes vasoconstriction, increased heart rate, high blood pressure, and

increased blood sugar level (Armitage & Hall, 1967; Hill, 1909; Thesleff, 1955). Regular

intake of nicotine may cause a broad spectrum of cancer diseases that affects several

body organs. Frequent detrimental health effects combined with the considerable

pervasiveness of cigarette smoke makes it a major cause of death worldwide (Alberg,

2008; Yildiz, 2004). Concentration as low as 30-60 mg for adults and 10 mg for children

are considered to be lethal (Cameron et al., 2014). Therefore, determination and

quantification of nicotine is an important analysis parameter that makes the basis of the

quality of the product in medicine and tobacco industry (Davis, 1986; Goniewicz, Kuma,

Gawron, Knysak, & Kosmider, 2013).

82

Several methodologies as well as analytical techniques such as high performance liquid

chromatography (HPLC) (Mahoney & Al-Delaimy, 2001; C. Wu, Siems, Hill, & Hannan,

1998), gas chromatography (GC) (Davis, 1986; Patrianakos & Hoffmann, 1979), liquid

chromatography-mass spectrometry (LC-MS) (McManus, deBethizy, Garteiz,

Kyerematen, & Vesell, 1990), immunochromatography (Gonzalez, Foley, Bieber,

Bourdelle, & Niedbala, 2011), spectrophotometry (Puhakainen, Barlow, & Salonen,

1987), capillary electrophoresis (CE) (Matysik, 1999), spectrofluorimetry, raman

spectroscopy (Mamián-López & Poppi, 2013), radioimmunoassay, electroanalytical assay

with different electrode systems have been reported for determination of nicotine

(Matysik, 1999; Suffredini et al., 2005; Švorc, Stanković, & Kalcher, 2014). Above-

mentioned analytical methods possess several advantages; nonetheless, the applicability

of these methods for routine analysis is impeded by tedious sample preparation

procedures for preliminary extraction and purification of nicotine that often leads to

sizeable loss of analyte and prolonged analysis time. Furthermore, hi-tech and expensive

instrumentation is required.

Linear-scan cyclic voltammetry are efficient techniques which were employed since

decades for generating mono / diradical(s) (i.e., anion or cations) and to study their

subsequent reactions. Cyclic voltammetry (CV) is perhaps one of the most practiced

electroanalytical technique owing to its simplicity of operation and versatility of

applications in diverse disciplines such as inorganic, organic and biochemistry

(Electroanalytical Methods - Guide to Experiments and Applications, 2010; Heinze,

1984; Kissinger & Heineman, 1983). Cyclic voltammetry allows for detailed study of rate

kinetics as well as thermodynamics of radical formation and their reaction. Beside these

obvious advantages, one of the foremost obstacle in investigating complex molecules and

or (bio) mixtures via cyclic voltammetry is limited sensitivity of conventional electrodes.

Moreover, the oxidation / reduction of nicotine and its metabolites requires extremely

large potential window which is generally out of the range of conventional electrodes

[22]. Therefore, modification of the electrode is often a prerequisite to enhance detecting

(sensing) efficiency of electrode.

83

Several reports on modification of glassy carbon electrode (GCE) for determination of

nicotine by cyclic voltammetry have been reported. A boron-doped diamond electrode to

improve the separation of nicotine peak in alkaline media using a potential window from

+0.6 to +1.8 V was reported by Suffredini et al (Suffredini et al., 2005). A screen printed

electrode modified by metallic free carbon nanotube cluster was employed for the

detection of nicotine in artificial saliva in a potential range of -0.4 to +1.2 V (Highton,

Kadara, Jenkinson, Logan Riehl, & Banks, 2009). The effects of thin-layer diffusion in

the electrochemical detection of nicotine on multi-walled carbon nanotubes modified

basal plane pyrolytic graphite (MWCNT-BPPG) electrode at a potential range of 0.0 to

+1.0 V was evaluated (Sims, Rees, Dickinson, & Compton, 2010). In another study,

nano-carbon was employed as an alternative to multi-walled carbon nanotubes in

modified electrodes using potential range of -1.5 to +1.5 V (Lo, Aldous, & Compton,

2012). Nicotine was determined in tobacco samples based on mussel-inspired reduced

graphene oxide-supported gold nanoparticles at a potential range of -0.2 to +0.6 V (Y.

Jing et al., 2016). In our recent publications, stabilization and applications of NPs by

homo and copolymers of P2VP has been demonstrated (Rahim et al., 2017; Rahim,

Khalid, Bhanger, Shah, & Malik, 2018).

In this study, poly(2-vinylpyridine-block-methyl methacrylate) [P2VP-b-PMMA or

P(2VP-MMA)] coated gold nanoparticles (AuNPs) modified glassy carbon electrode,

[P(2VP-MMA)-AuNPs]-GCE, is used as a valuable tool to study the electrochemical

behavior of nicotine in aqueous as well as in organic media. P(2VP-MMA)-AuNPs were

synthesized and characterized by UV-Vis, FTIR, AFM and zetasizer. Further, the

prepared P(2VP-MMA)-AuNPs are coated on GCE for preparation of [P(2VP-MMA)-

AuNPs]-GCE. Polymer based nanoparticles-modified electrode offers high effective

surface area, enhanced mass transfer and better control over local microenvironment. The

large effective surface area results in more active sites and higher signal to noise ratio.

Higher rate of mass transport to the electrode surface is expected due to smaller

dimensions of nanoparticles. Furthermore, potential window of the bare gold should be

reduced by coating of P(2VP-MMA)-AuNPs. To the best of our knowledge, current study

is perhaps the first report on the enhancement of electroanalytical response for nicotine

by employing [P(2VP-MMA)-AuNPs]-GCE.

84

4.1 Experimental Section

4.1.1 Materials

Poly(2-vinylpyridine-block-methyl methacrylate) [P2VP-b-PMMA or P(2VP-MMA)]

block copolymers of various molar masses were purchased from polymer standards

services (Mainz, Germany). The specifications of the products as provided by the

manufacturer are listed in Table 4.1.

TableTetrachloroauric (III) acid trihydrate>99.9 % (HAuCl4.3H2O) (Sigma Aldrand ich,

USA) was used for the synthesis of gold nanoparticles. NaBH4>95.0% (TCI, Tokyo,

Japan), and HPLC grade solvents such as methanol >99.9%, and toluene > 99.9% (RCI

Labscan Limited, Thailand) were used as received.

Deionized water (DIW) was taken from ICCBS distillation plant. Acetonitrile (ACN)

>99.99% (Fischer scientific, USA) was dried over 3 Å molecular sieves to remove the

traces of water prior to use. Standard nicotine ≥ 99.0 % was purchased from Fluka

Chemie Gmbh (Buchs, Switzerland). Two supporting electrolytes (SE), tetra-n-butyl

ammonium perchlorate (TBAP) >99.0% (TCI, Tokyo, Japan) and potassium chloride

(KCl) >99.0 % (Merck, Germany), were used for non-aqueous and aqueous medium,

respectively. Silver nitrate (AgNO3) > 99.99% (Scharlau, Europe) was used for the

preparation of Ag/Ag+ reference electrode. All electrochemical experiments were carried

out at ambient room temperature (i.e. 28±1℃)

4.1.2 Instrumentation

UV–visible spectra were recorded with a double beam Shimadzu UV-1800 series

spectrophotometer (Kyoto, Japan) operated at 1 cm path length quartz cuvette. The

wavelength range from 190 to 800 nm was used.

The FTIR spectra were recorded on a Bruker Vector 22 spectrometer (Germany) by KBr

pellet method. Analyses of all samples were performed in the range of 400-4000 cm-1

.

Ten scans were recorded in order to obtain the spectral resolution of 0.1 cm–1

.

85

The size of P(2VP-MMA)-AuNPs were determined by atomic force microscopy (AFM)

using an Agilent 5500 microscope (Arizona, USA), equipped with triangular silicon

nitride cantilever (Veeco, model MLCT-AUHW) with a spring constant of 0.01 Nm-1

and

0.1 Nm-1

, operated in a tapping mode. Samples were prepared by putting a drop of freshly

prepared solution on a surface of silicon (Si) wafer, and dried in air.

Particle size distribution and zeta potential of P(2VP-MMA)-AuNPs were determined by

zetasizer, Nano-ZSP (Malvern Instruments). The analyses were performed at a scattering

angle of 90° using disposable cuvette for zetasizer and dip cell cuvette for zeta potential

studies, at 25 °C.

The electrochemical experiments were performed on CHI-600 series electrochemical

analyzer using CHI-600C software. Three electrode assembly (from CH Instruments,

Inc.) along with an electrochemical cell with teflon cap having five taper holes, was used

to record CVs. Working, reference and counter electrodes supplied by CH Instruments

Inc were fitted in these three holes prior to scan. Glassy carbon electrode (GCE; CHI104)

with an area of 0.070 cm-2

. Modified GCE (modification protocol discussed in later

section 2.3.2) was used as a sensor in combination with Ag/AgCl (CHI111) and Ag/Ag+

(CHI112) as a reference electrode in aqueous and non-aqueous media, respectively.

Platinum wire (CHI115) was used as a counter electrode.

Table 4.1. Molecular weight and polydispersity index of P(2VP-MMA), as provided by

manufacturer

Sample Mn

(g/mol)

Mw

(g/mol)

Mp

(g/mol)

PDI

Mw/ Mn

Percent Ratio

(P2VP:PMMA)

P(VP3-MMA97) 23300 69200 59700 2.69 3:97

P(VP15-MMA85) 28300 47300 52600 1.67 15:85

P(VP10-MMA90) 40400 149000 221000 3.66 10:90

Where Mn, Mw, Mp and PDI are number average molar mass, weight average molar

mass, molar mass at peak maximum, and polydispersity index respectively. Subscripts in

sample coding represent the percent ratio of both blocks.

86

4.1.3 Methods

4.1.3.1 Preparation of P(2VP-MMA)-AuNPs

Synthesis of P(2VP-MMA)-AuNPs was accomplished using a two-phase one pot system

consisting of toluene and methanol (90:10), Figure 4-1. Polymers form spherical micelles

in toluene where polar poly(2-vinylpyridine) (P2VP) segment makes the core while

comparatively non-polar poly(methyl methacrylate) (PMMA) segment extends outward

making shell. 1.0 mL (0.1 mM) aliquot of P(2VP-MMA) solution was added into 20 mL

(0.25 mM) solution of HAuCl4.3H2O. The resulting solution was subsequently stirred for

30 min, allowing the [AuCl4]-1

to diffuse into the core of the micelles and making a

complex with the pyridine groups of P2VP. 0.1 mL of 16 mM NaBH4 solution were

added into the reaction mixture that reduce the Au (III) into Au (0) (Deraedt et al., 2014).

The appearance of pink color is the indication of formation of gold nanoparticles.

Figure 4-1: Schematic illustration of the reduction process of Au (III) particles in the

presence of a stabilizing block copolymer P(2VP-MMA) using NaBH4 as reducing agent.

87

4.1.3.2 Electrochemical studies

For electrochemical study of AuNP on nicotine determination GCE was modified with

selected AuNps. Modification of GCE was carried out by adopting a simple drop cast

procedure. This was done by adding 1-2 drops of P(2VP-MMA)-AuNPs on surface of

GCE and left it as an upward position to air dry for least 7-10 min. After complete

drying, modified GCE was used as a sensor in connected with reference and counter

electrode. As modification, renewing surface of GCE is also a staple part of this

electrochemical study presented here. Hence, after each scan the surface of working

electrode, P(2VP-MMA)-AuNPs-GCE, was renewed by polishing it with alumina (mesh

size 0.3 micron) and sonicated in acetone followed by distilled water for 5 min.

A blank test solution was prepared by taking 5.0 mL of 0.1M solution of SE (KCl or

TBAP) in electrochemical cell for voltammetric titration. This solution was then titrated

by stepwise addition of specific volume (μL) of freshly prepared 0.1-0.2 M nicotine to

maintain the minimum concentration and scan voltammogram. All voltammetric

measurements were referred to Ag/AgCl and / or Ag/Ag+ with the scan rate of 0.1 Vs

-1.

For non-aqueous system, silver-silver ion (Ag/Ag+) electrode was used with freshly

prepared 5.0 mM solution of AgNO3 and 0.1M tetra-n-butyl ammonium perchlorate in

acetonitrile.

Voltammetric (oxidative) wave of nicotine appears at positive potential, though an

attempt of degassing the test solution by bubbling high purity argon gas is to be an

optional. Thus, deoxygenate when scan towards negative potential direction only.

Scanning potential (positively) from 0 to +1.4V and subsequently reversing it back

remained as a usual practice throughout this study.


4.2.1 Characterization of P(2VP-MMA)-AuNPs

Reduction of gold ions into gold nanoparticles is indicated by conversion of yellow

reaction mixture to pink, also confirmed by measuring UV–vis spectra of the solution that

88

showed an absorption band at 525 nm (Figure 4-2). Typically, gold nanoparticles have an

characteristics absorption band between 500 and 600 nm (Frederix et al., 2003).

Figure 4-2: UV-visible spectra of P(2VP-MMA)-AuNPs stabilized by different block

copolymers varying in total molar mass and chemical composition

The formation of P(2VP-MMA)-AuNPs was attributed to the coordination of lone pair of

electrons on nitrogen of pyridine ring in the polymer with gold particles. The comparison

of FTIR spectra of P(2VP-MMA), P(2VP-MMA)-AuNPs and AuNPs supports the

assumption (Figure 4-3). The absorption bands at 1150 cm-1

and 1247 cm-1

are attributed

to the C-O-C stretching vibration, and two bands at 1388 cm-1

and 752 cm-1

to the α-

methyl group vibrations in PMMA. The bands at 1065, 985 and 843 cm

-1 are the

characteristic peaks for vibration of PMMA. The 1732 cm-1

band shows the presence of

the acrylate carboxyl group. The band at 1444 cm-1

can be assigned to the bending

vibration of C-H bonds of the -CH3 group and the two bands at 3002 cm-1

and 2952 cm-1

to C-H bond stretching vibrations of the -CH3 and -CH2- groups, respectively.

Furthermore, two weak absorption bands at 3442 cm-1

and 1641 cm-1

can be assigned to

the stretching and bending vibrations of –OH group of absorbed moisture, respectively.

89

The characteristic -C=N pyridine ring vibrations appear at 1595 cm-1

. The disappearance

of -C=N band and the appearance of 1650 cm-1

band after loading and reduction of

AuNPs may be due to the fact that P2VP block in the core of P(2VP-MMA) micelles is

mainly responsible for the reduction of Au (III) ions.

Figure 4-3: Comparative FTIR spectra of P(2VP-MMA)-AuNPs, P(2VP-MMA)

and AuNPs

The reduction activity of P2VP hompolymers increases with increase in the molar mass

(Rahim et al., 2017). On the same lines, we expect effect of the molar mass of P2VP

block in the block copolymer on the reduction reactivity. The spherical shape and

localized nature of P(2VP-MMA)-AuNPs was also confirmed by AFM. Moreover, the

block copolymers make micelles with P2VP core shielded by PMMA shell, Figure 4-4.

90

Figure 4-4: AFM images of P(2VP-MMA)-AuNPs

Furthermore, the particles size and distribution of AuNPs depends upon both the total

molar mass and molar mass of P2VP block. The average size and size distribution

profiles of P(2VP3-MMA97)-AuNPs, P(2VP15-MMA85)-AuNPs, and P(2VP10-MMA90)-

AuNPs as analyzed by zetasizer are shown in Figure 4-5. The P(2VP3-MMA97) and

P(2VP15-MMA85) have similar total molar mass, however, the length of P2VP block of

latter is higher. The average diameter of the P(2VP3-MMA97)-AuNPs and P(2VP15-

MMA85)-AuNPs are 84.90 ± 46.72 nm and 140.5 ± 80.7 nm, respectively. On the other

hand, the average diameter of P(2VP10-MMA90)-AuNPs (151 ± 98.59 d.nm) is even

higher than the P(2VP15-MMA85)-AuNPs (140.5 ± 80.7 nm). In this case, the length of

P2VP block is similar; however, total molar mass of the latter is lower. Therefore, we can

conclude that the size of the AuNPs prepared by P(2VP-MMA) increases with the molar

mass of individual P2VP block as well as with the total molar mass of the block

copolymer.

91

Figure 4-5: Size distribution by intensity of P(2VP3-MMA97)-AuNPs, P(2VP15-MMA85)-

AuNPs, and P(2VP10-MMA90)-AuNPs.

Zeta potential indicates the presence of positive charges on the surface of P(2VP-MMA)-

AuNPs. The zeta potential of P(2VP3-MMA97)-AuNPs, P(2VP15-MMA85)-AuNPs, and

P(2VP10-MMA90)-AuNPs are given in Figure 4-6. The surfaces charges on P(2VP3-

MMA97)-AuNPs, P(2VP15-MMA85)-AuNPs, and P(2VP10-MMA90)-AuNPs were 23.5,

0.0276 and 0.0196 mV, respectively. More positive charge on P(2VP3-MMA97)-AuNPs

can be noticed compared to other two samples. The reason might be better interaction of

P2VP with AuNPs because of stable micelles of P(2VP3-MMA97) in toluene owing to its

larger PMMA block. The positive charges on the polymer surface make the P(2VP-

MMA)-AuNPs electroactive in nature, that reacts with the basic nitrogen atoms in

nicotine. This interaction allowed for employing the P(2VP3-MMA97)-AuNPs as novel

electrochemical sensor for nicotine. P(2VP3-MMA97)-AuNPs seems to be more stable

compared to other two polymer combinations with Au and have high surface charges,

hence, might acts as a better sensor for nicotine. Therefore, P(2VP3-MMA97)-AuNPs are

selected for further studies.

92

Figure 4-6: Zeta potential distribution P(2VP3-MMA97)-AuNPs, P(2VP15-MMA85)-

AuNPs, and P(2VP10-MMA90)-AuNPs

As a next step, the stability of the synthesized AuNPs as a function of storage time was

monitored by UV-Vis spectroscopy and AFM analysis. The P(2VP3-MMA97)-AuNPs

remained stable for fairly longer time at ambient temperature, Figure 4-7. UV-Vis

spectroscopy reveals the enhancement in the SPR signal with storage time upto two

months, Figure 4-7 A-supplementary material. However, SPR signal decreased after two

months that indicates the agglomeration of the NPs into aggregates. These observations

vis a vis stored AuNPs at different time interval were further confirmed by AFM analysis

(Figure 4-7 B).

93

Figure 4-7: Time stability of P(2VP3-MMA97)-AuNPs (A) UV visible spectroscopy (B)

AFM. All the images are of 2x2µm

Thermal stability of P(2VP3-MMA97)-AuNPs was evaluated by elevating temperature of

specified polymer solution to 100 °C for 10 minutes and then cooling it to ambient

temperature (25 °C). UV-vis spectrum of temperature treated sample showed an

enhancement in absorption maxima (Amax) of SPR band which indicates relatively higher

stability. At higher temperatures, solubility of polymers increases that provides larger

functionalized area and promotes conversion of gold ions into gold nanoparticles thus

enhance the stability of AuNPs (Figure 4-8) (Rahim et al., 2017).

94

Figure 4-8: Temperature effect on P(2VP3-MMA97)-AuNPs

Generally, addition of electrolytes in NPs solution results in aggregation of nanoparticles.

A thorough study was carried out to elucidate the effect of concentration of electrolytes

(0.001mM – 5M NaCl) on P(2VP3-MMA97)-AuNPs stability, monitored by UV-visible

spectroscopy. P(2VP3-MMA97)-AuNPs were stable over a wide range of concentration of

sodium chloride, Figure 4-9.

95

Figure 4-9: Electrolyte effect on the stability of P(2VP3-MMA97)-AuNPs

Effect of pH on the stability of P(2VP3-MMA97)-AuNPs was examined in a range of 2-12

by monitoring the change in SPR band in UV-visible spectrum, Figure 4-10. No change

in position and shape of the SPR band was observed with variations in pH from 2 to 12,

confirming that aggregation and agglomeration is protected over a wide range of pH. The

enhancement in SPR band at higher pH might be due to formation of lesser reactive

[AuCl(4-n)(OH)n]- complex by the reaction of [AuCl4]

- with OH

-, where n increases with

increase in the pH (Badawy et al., 2010a; Gandubert & Lennox, 2005c; Tyagi et al.,

2011b).

96

Figure 4-10: pH effect on P(2VP3-MMA97)-AuNPs

4.2.2 Cyclic Voltammetric detection of nicotine using P(2VP3-MMA97)

-AuNPs-GCE as a Sensor

Cyclic voltammetry (CV) was used to explore the electrochemical sensing application of

the prepared nanocomposites (P(2VP3-MMA97)-AuNPs, (P(2VP15-MMA85)-AuNPs and

P(2VP10-MMA90)-AuNPs) for the detection of nicotine. In this context, glassy carbon

electrode (GCE) was modified with above mentioned composites (polymer stabilized

AuNPs) for its application as a redox probe. Nonetheless, this work primarily emphasizes

on P(2VP3-MMA97)-AuNPs. The reason for emphasizing specifically on P(2VP3-

MMA97)-AuNPs is; a well-defined oxidative wave of nicotine with significant peak

intensities (current response) via [P(2VP3-MMA97)-AuNPs]-GCE was observed, Figure

4-11.

97

Figure 4-11: Voltammetric response of nicotine on (a) bare GCE; (b) P(2VP3-MMA97)-

AuNPs, (c) P(2VP15-MMA85)-AuNPs, and (d P(2VP10-MMA90)-AuNPs. fabricated GCE.

It could infer as, the electron conductivity (or tunneling) through [P(2VP3-MMA97)-

AuNPs]-GCE is higher compared to the other composites modified GCE probes. Hence,

the modification of GCE offers an enhanced electrochemical area to electrode which

facilitates the electron transfer kinetic between the surface of P(2VP3-MMA97)-AuNPs-

GCE and nicotine. The appearance of an irreversible peak in the anodic region at

voltammetric time scale as a result of electro-oxidation of nicotine is an established fact.

By scanning potential anodically, 0 to +1.4V, no voltammetric peak / wave appeared at

the bare GCE in the absence of nicotine (both in aqueous and non-aqueous medium),

Figure 4-12. Beside this, a completely irreversible peak appeared at +1.154V at NPs

composite-modified GCE in the absence of nicotine while acetonitrile was used as a

solvent, Figure 4-13.

98

Figure 4-12: Cyclic voltammograms in the absence of nicotine on bare GCE while using

(a) acetonitrile (b) water as a solvent.

Figure 4-13: A comparative view of cyclic voltammograms (a) absence (0 mM) and (b)

presence (0.05 mM) of nicotine on P(2VP3-MMA97)-AuNPs-GCE in acetonitrile.

99

This redox peak seems to be a characteristic peak of the composite, P(2VP3-MMA97)-

AuNPs sensor, applied for GCE fabrication. Nevertheless, in aqueous medium at pH =

6.8 ± 0.1, an ill-defined wave or a slight hump (of composite) appeared at +1.02V (Figure

not shown). The electro inactive nature of P(2VP3-MMA97) was confirmed by casting

pure P(2VP3-MMA97) on GCE surface in acetonitrile, where no peak appeared in anodic

region, Figure 4-14.

Figure 4-14: An overlay of (a) absence, (b) and (c) presence of nicotine on P(2VP3-

MMA97)-GCE in acetonitrile at scan rate of 0.1V.s-1

.

Therefore, suggesting that the peak appeared at +1.154V is perhaps due to P(2VP3-

MMA97)-AuNPs. Unstable AuNPs (in absence of polymer) does not show any

appreciable electrochemical response for nicotine. Scanning potential in negative region

of deoxygenated solution (of 0.1 M TBAP / KCl) was also attempted for the peak

referred to sensor and no peak or hump was observed.

100

Nicotine is an alkaloid, more soluble in polar-aprotic solvents compared to aqueous

medium. The difference could clearly be observed in the voltammograms depicted in

Figure 4-15 A,B. The bare GCE sensor responds to nicotine concentration higher than

0.2M in acetonitrile, Figure 4-15 A. Conversely, only slight or ill-defined humps

appeared in aqueous medium (see Figure 4-15 B). Nonetheless, this study significantly

focuses on non-aqueous medium i.e. acetonitrile. Same GCE after coating with P(2VP3-

MMA97)-AuNPs was tested for its electrochemical response for nicotine. Thereafter, the

selectivity of P(2VP-MMA)-AuNPs-GCE sensor as a function of nicotine in acetonitrile

was investigated. The current density increased with increase in the concentration of

nicotine as shown in Figure 4-16. Well-defined concentration dependent cyclic

voltammetric peaks appeared on modified GCE. The detection limit of the modified GCE

sensor is enhanced, down to 0.1 mM nicotine concentration.

Comparative view of electrochemical response of nicotine on bare GCE, P(2VP3-

MMA97)-GCE, P(2VP3-MMA97)-AuNPs-GCE is demonstrated in Figure 4-17.

101

Figure 4-15: Cyclic voltammograms of nicotine with various concentrations ranging from

0.05 mM to 0.4 mM on bare GCE in (A) acetonitrile; (B) distilled deionized water.

Figure 4-16. Effect of various concentrations (from 0.05 mM – 0.4 mM) of nicotine on

P(2VP3-MMA97)-AuNPs-GCE in acetonitrile.

102


and (b) P(2VP3-MMA97)-AuNPs-GCE and (c) P(2VP3-MMA97)-GCE-sensor in

acetonitrile.

Highest sensitivity for 0.4mM nicotine is obtained with P(2VP-MMA)-AuNPs-GCE. The

P(2VP3-MMA97)-GCE does not show any response while response of bare GCE is

considerably less compared to P(2VP3-MMA97)-AuNPs-GCE. Figure 4-18 shows the

electrochemical response of 0.1 mM nicotine on bare GCE and P(2VP3-MMA97)-AuNPs-

GCE, in acetonitrile.

103


and (b) P(2VP3-MMA97)-AuNPs-GCE in acetonitrile.

As can be noticed, bare GCE was not able to detect nicotine compared to an effective

response shown by P(2VP3-MMA97)-AuNPs-GCE. Hence, the detection limit and

efficiency of modified GCE is improved considerably compared to bare GCE for

nicotine. The comparison of peak current as a function of nicotine concentration is

demonstrated in Figure 4-19.

Figure 4-19: Plot of oxidative peak current as a function of concentration of nicotine (0.1

mM - 0.5 mM).

104

The peak current increases with increasing concentration of nicotine linearly with a high

statistical correlation R2

value, 0.986. The stability and reproducibility of nicotine sensor

P(2VP3-MMA97)-AuNPs-GCE was also investigated at different time intervals. For this

particular study, the sensor was refrigerated (below 5°C) for more than 4 weeks and

evaluated for its performance.

4.3 Conclusion

In this study, a novel P(2VP3-MMA97)-AuNPs-GCE based electrochemical sensor is

reported for rapid quantitative assay of nicotine. Stability and homogeneous nature of

P(2VP3-MMA97)-AuNPs was confirmed by UV-Vis, FTIR, AFM, and zetasizer. The

sensitivity of bare GCE is significantly enhanced by coating with P(2VP3-MMA97)-

AuNPs. A well-developed voltammetric peak appeared at +0.66 V (versus Ag/Ag+), in

acetonitrile for determination of nicotine in the concentration range of 0.1 – 0.4 mM with

a detection limit of 0.16 mM. The P(2VP3-MMA97)-AuNPs-GCE is more sensitive

towards nicotine, the electrochemical response obtained by P(2VP3-MMA97)-AuNPs-

GCE is enhanced by an enhancement factor of ~2 compared to bare GCE. Simple, facile,

low cost synthesis and high stability of P(2VP3-MMA97)-AuNPs-GCE make it a valuable

choice for routine laboratory testing.

105

Chapter 5

Selectivity of thin films of poly(2-vinylpyridine-block-

methyl methacrylate) copolymers: an AFM study

106

Abstract

The surface morphologies of poly(2-vinyl pyridine-block-methyl methacrylate) (P2VP-b-

PMMA) (P(2VP-MMA)) copolymer thin films were analyzed via atomic force

microscopy. Different morphologies were observed with different molecular weights and

compositions of P(2VP-MMA). Moreover, the incorporation of gold nanoparticles

greatly influenced the surface morphology of P(2VP-MMA) and alter its surface

properties. The morphology of P(2VP-MMA) copolymer thin films were different from

solvent to solvent; for films cast from toluene, the poly(methyl methacrylate) (PMMA)

phase appeared as pits in the P2VP matrix, whereas the thin films cast from chloroform

solution exhibited a melted structure and small, separated PMMA phases as protrusions

over the P2VP was appeared in ethyl acetate. The annealing temperature affected the

surface morphology of P(2VP-MMA) copolymer thin films; the poly(2-vinyl pyridine)

(P2VP) phases at the surface were increased when the annealing temperature was higher

than the P2VP glass-transition temperature. The microphase structure of P(2VP-MMA)

copolymer thin films were also strongly influenced by different substrates.

5 Introduction

Block copolymer (BCP) films offers imaginable self-organized patterned morphologies

of molecular dimensions in a highly effective way due to difference between

compatibility and thermodynamic properties of different blocks. This high level of

control over nanostructure morphologies is required while working for miniaturization of

electronic and optical devices (Campoy-Quiles et al., 2008; G. Li, Shrotriya, Huang, et

al., 2005; Ma, Yang, Gong, Lee, & Heeger, 2005). Depending on the length,

connectivity, and mutual interactions of the different blocks, the microdomains can form

spherical, lamellar, cylindrical, gyroid, or more complex shapes that exhibit regular

periodic order with typical repeat distances in a range of 10-100 nm. Various factors that

affect the surface morphology of thin films of polymers include molecular weight and

composition of block copolymer, casting solvent, annealing temperature, film thickness,

107

interfacial interactions, solvent evaporation, substrate pattern, and electric fields (Cui,

Ding, Li, Wang, & Han, 2006; Paeng, Richert, & Ediger, 2012; Roy & Sharma, 2015).

The prepared films exhibit a laterally different but highly ordered distribution of different

polymeric components with microdomain sizes and characteristics distances at the

nanoscale. They have been used as self-organized templates for synthesis of various

inorganic materials such as nanoparticles, nano-clusters, nanotubes, nanowires etc. The

surface structure and morphology are important aspects of block copolymers, and they

are determined by a minimization of the surface and interfacial energy. On chemically

homogeneous surfaces, differences in the interfacial energy between the surface and the

blocks of the copolymer generally induce different surface morphologies of the film to

minimize the free energy. In the bulk, the mesoscale structure of BCPs is determined by

molecular parameters, such as chain length, volume fractions of the components, degree

of incompatibility, and temperature. However, some additional driving forces exist for

structure formation in thin films. Typically, polymeric components with the lowest

surface energy will preferentially accumulate at the surface and the component with

lowest interfacial energy will be attracted to the supporting substrate. Furthermore,

confinement of the material to a film thickness that is a non-integer multiple of the

―natural‖ bulk repetition length can cause the thin film structures to deviate from the

corresponding bulk material. As a result, the phase behavior in thin film of block

copolymers is more complex and exhibits a larger variety of structures compared to

found in the bulk. Many approaches are used to alter the self-organization of BCPs in

which thermal annealing is one of the most advanced approaches to modify the polymer

structure at nano-scale. Generally, post deposition of polymer onto the substrate lead

disordered structure. The polymers become mobile and structurally transform above the

glass transition temperature (Tg). Thermally induced molecular motion reorganizes

amorphous portion into more perfect crystalline form, while bulk melting, which destroys

existing crystalline regions. Therefore, maximum crystallinity can only be achieved

below Tm (Jo, Kim, Na, Yu, & Kim, 2009; G. Li, Shrotriya, Yao, & Yang, 2005;

Verploegen et al., 2010; N. Wu, Zheng, Huang, & Liu, 2007).

108

Block copolymers composed of poly(2-vinylpyridine) (P2VP) segment, contain pyridine

moieties as side chains, have been used for many industrial applications. A typical

example is utilization of P2VP block copolymers as templates for metal complexes to

prepare nanoparticles (NPs). The NPs-P2VP based nanocomposite assemblies improve

the functionality of electronic, photonic, and chemical devices due to the presence of

nitrogen atoms of the pyridine ring with an unshared electron pair that induces pH

sensitivity (N. Wu et al., 2007). In addition, magnetic, photonic, chemical, and electrical

properties of nanomaterials are very different from the bulk materials. Consequently,

there have been several studies focused on the incorporation of nanoparticles within the

block copolymer microdomains (Carotenuto et al., 2000; Lekesiz et al., 2015; Nasir,

Kausar, & Younus, 2015; Youk et al., 2002; Yu et al., 2008).

The shape and size of the patterns of thin films obtained by block copolymers are highly

dependent upon the total molar mass, molar mass of individual blocks and chemical

composition of parent materials. In this study, poly(2-vinylpyridine)-block-poly(methyl

methacrylate) [P2VP-b-PMMA, P(2VP-MMA)], an amphiphilic block polymer, is

utilized for preparation of micelles in suitable media. Atomic force microscopy (AFM)

being a non-destructive imaging technique is employed for structural and morphological

analysis of the thin films and gold nanoparticles incorporated into BCPs.

5.1 Experimental


Poly(2-vinylpyridine-block-methylmethacrylate) [P(2VP-MMA)] block copolymers were

purchased from polymer standard services (Mianz, Germany). Tetrachloroauric acid

(HAuCl4) (Sigma Aldrich, USA) was the starting material for the synthesis of gold

nanoparticles. NaBH4 (TCI, Tokyo, Japan) was used as reducing agent for HAuCl4.

HPLC grade toluene, chloroform, and ethyl acetate (RCI Labscan limited, Thailand) were

used as solvents. All the reagents were used as received. The molecular weights and their

distributions (PDI) of the block copolymers are summarized in Table 5.1.

109

Table 5.1: Molecular weight and polydispersity index of P2VP-b-PMMA as provided by

manufacturer

Sample Mn

(g/mol)

Mw

(g/mol)

Mp

(g/mol)

PDI

Mw/ Mn

Percent ratio

(P2VP:PMMA)

P(VP3-MMA97) 23300 69200 59700 2.69 3:97

P(VP15-MMA85) 28300 47300 52600 1.67 15:85

P(VP10-MMA90) 40400 149000 221000 3.66 10:90

Where Mn, Mw, Mp and PDI are number average molar mass, weight average molar

mass, molar mass at peak maximum, and polydispersity index respectively. Subscripts in

sample coding represent the percent ratio of both blocks.

5.1.2 Atomic Force Microscopy

AFM images of P(2VP-MMA) and P(2VP-MMA)-AuNPs were recorded by Agilent

5500 atomic force microscope (AFM), (Arizona, USA). Triangular soft silicon nitride

cantilever (PPP-NCH) with a length of 125 µm, thickness of 4 µm and a mean width of

30 µm, having a spring constant value of 42 Nm-1

, in the tapping mode was used for all

measurements. A resonance frequency in the range of 204–330 KHz was used; resonance

peaks typically at 307 KHz in the frequency response of the cantilever were chosen for

the tapping- mode oscillation. The AFM images were obtained with a maximum scan

range of 10x10 µm; the scanning frequencies were 1.01 Hz/line. The measurements were

carried out in insulated chamber under hanging position and weightless conditions.

5.1.3 Sample Preparation

P(2VP-MMA) copolymers were dissolved in toluene to make 0.1 mM solutions, and thin

films were obtained by the spin coating of a solution of P(2VP-MMA) copolymer in

toluene at room temperature onto various substrates at 4000 rpm for 30 s, which had an

atomically smooth surface. Spin-coated films from chloroform and ethyl acetate solutions

of P(2VP-MMA) copolymer were prepared following similar procedure.

110


Surface chemistry of polymer with desired physical characteristics such as size, shape,

and interfacial features are the main rationale of many applications in various fields while

working at nanoscale. Poly(2-vinylpyridine-block-methylmethacrylate) (P(2VP-MMA))

is an amphiphilic diblock copolymer. The structure of polymer is given in Figure 5-1.

Figure 5-1: Structure of poly(2-vinylpyridine-b-methylmethacrylate)

BCPs have the ability to self-assemble in the form of spherical micelles in a suitable

medium. For example, toluene is a good solvent for PMMA and non-solvent for P2VP,

therefore, the micelles are formed with insoluble hard core containing P2VP and

soluble PMMA corona (Figure 5-2). The presence of basic nitrogen in the P2VP

matrix of copolymer makes it an excellent candidate for fabricating nanoparticles in

the polymer domain.

111

Figure 5-2: Schematic representation of micellization and self-organization of AuNPs in

P2VP domain

This study is divided into four parts. First, we discuss the surface morphology of the

copolymer thin films with different compositions, molecular weight and effect of

incorporation of nanoparticles in polymer matrix. The second part includes effect of

different solvents on the morphology of film structure. Third section include, effect of

polymer–substrate interaction on the film morphology, while the effect of thermal

annealing on the morphology of the copolymer films will be discussed in the fourth

section.

112

5.2.1 Characterization of Surface Morphology

Polymer and polymer coated AuNPs solutions of 0.1 mM in toluene were casted onto Si

wafer. Surfaces were placed for 48 hrs in order to evaporate the solvent. Figure 5-3A-B

illustrate, surface morphology of thin films obtained on Si wafer by casting P(2VP-

MMA) varying in molar mass and compositions of individual blocks. As can be noticed,

different surface topographies were obtained that require quantitative assessment for

development of any relation with the composition of polymers. Three factors are

involved in controlling the phase separation of copolymer in formation of thin films by

evaporation of the solution: (1) different surface free energies of polymer, (2) solubility

factor in common solvent and (3) polymer–air and polymer–substrate interactions. All

three factors collectively dictate the final morphology of copolymer films. Importantly,

incorporating gold nanoparticles affected the topography of the polymer by enlarging

phase domain, Figure 5-2.

BCPs can be compared by varying two parameters. Firstly, BCP having similar molar

mass but varying composition of both blocks is compared. In this context, P(VP3-

MMA97) and P(VP15-MMA85) have similar total molar mass, nonetheless, the mole

fraction of both blocks vary significantly. Flatter surfaces with lamellar structure having

undefined grooves between lamellea was obtained by P(VP3-MMA97) because the block

length of P2VP is smaller compared to PMMA, therefore, they arrange like threads

instead of spherical micelles. While, continuous hexagonal structures having a pore

size 28 nm were obtained with P(VP15-MMA85), Figure 5-3 A. Furthermore, P(VP15-

MMA85) has similar block length of P2VP compared to P(VP10-MMA90) while total

molar mass of later is higher. Thin film obtained by P(VP10-MMA90) is similar in basic

morphology with the P(VP15-MMA85), however, smaller cylindrical nanogrooves with

the average pore size of 10 nm are obtained, Figure 5-3 B.

113

Figure 5-3: AFM topographical images of P(2VP-MMA) with schematic overview of the

fabrication of nanoporous layers by P(2VP-MMA) (A) P(2VP-MMA) copolymers with

different compositions (sample area, 10x10 µm) (B) P(2VP-MMA) copolymers with

different molecular weights (sample area, 10x10 µm). The scale bar on each image show

2.5 µm.

The grooves and ridges observed in images might be pores of the block copolymers

formed during the process of micelles formation. The hypothesis is supported by

comparison of topographical images obtained by P(2VP-MMA) and P(2VP-MMA)-

AuNPs, Figure 5-3 A-B and Figure 5-4 A-B. The nanoparticles incorporated in the

polymer micelles become brighter brighter and enlarge the domain size. The basic

nitrogen in the polymer backbone reacts with AuNPs that result in the swelled core of

the micelle. It can be noticed that the size of the nanoparticles fabricated with different

copolymer is in order of P(VP10-MMA90) < P(VP15-MMA85) < P(VP3-MMA97). This is a

clear indication that pores in P(VP10-MMA90) are smaller compared to P(VP15-MMA85),

followed by P(VP3-MMA97).

114

Figure 5-4: Morphology of P(2VP-MMA)-AuNPs

Further, the obtained Rsk values for P(VP3-MMA97), P(VP15-MMA85) and P(VP10-

MMA90) are 1.55, 3.28 and 2.08, respectively. These values clearly indicate the

unsymmetrical surfaces containing grooves. Lowest value of Rsk for P(VP3-MMA97)

compared to other two BCPs indicate comparatively smoother surface. Increase in the

mole fraction of P2VP with similar molar mass resulted in higher Rsk values that

means rough surface, compare P(VP3-MMA97) and P(VP15-MMA85). Furthermore,

slightly lower Rsk values obtained for P(VP10-MMA90) compared to P(VP15-MMA85)

might be attributed to longer PMMA block with similar P2VP block length, Figure 5-5A.

The roughness of the film decreases by incorporation of gold nanoparticles as is

evident with decreased Rsk values, Figure 5-5B.

115

Figure 5-5: Amplitude roughness profile of (A) P(2VP-MMA), and (B) P(2VP-MMA)-

AuNPs at horizontal scale of 10x10 µm.

The disparity of the morphology of thin films for P(2VP-MMA) and P(2VP-MMA)-

AuNPs might be attributed to a distinct conformation with the different surfaces. Polymer

roughness factors are quantitatively evaluated for both P(2VP-MMA) and P(2VP-MMA)-

AuNPs by analysis of the AFM images. The roughness root mean square (RMS) was

calculated at 10 µm length scales. As can be noticed by the phase images that P(VP15-

MMA85) was found to have relatively rough surface, with an RMS value of 71.6 nm at 10

µm, Figure 5-6. Smoother surfaces of thin films obtained by P(VP3-MMA97) and P(VP10-

MMA90) are evident with RMS values of 14.4 and 19.3 nm respectively.

116

Figure 5-6: AFM 3D phase images of P(2VP-MMA) and P(2VP-MMA)-AuNPs

showing the thickness of the film on the Si wafer. From top to the bottom: P(VP3-

MMA97), P(VP15-MMA85), P(VP10-MMA90) (left); P(VP3-MMA97)/AuNPs, P(VP15-

MMA85)/AuNPs and P(VP10-MMA90)/AuNPs, (right)

Incorporation of AuNPs resulted in a decrease in the RMS values. The decrease is very

pronounced by a factor of 6.2 for P(VP15-MMA85) that has comparatively roughest film

surface. Other two polymers namely P(VP3-MMA97) and P(VP10-MMA90) show a

reduction in RMS values by a factor of 1.04 and 1.56, respectively. The comparison of

reduction in RMS values by incorporation of AuNPs is demonstrated in Figure 5-7.

P(VP15-MMA85) has been selected for further study of effects of various experimental

parameters on the morphology thin film.

117

Figure 5-7: Comparison of P(2VP-MMA) and P(2VP-MMA)-AuNPs RMS values

obtained through AFM studies

5.2.2 Effect of Casting Solvent

The casting solvent has a huge impact on the morphology of block copolymer films

(Campoy-Quiles et al., 2008; Verploegen et al., 2010; N. Wu et al., 2007). The phase

exhibiting a lower solubility in the solvent used for casting extends beyond the other

phase with higher solubility in the copolymer. Solvent effects with different solubility

and selectivity values on the surface morphology of copolymer films have been studied to

investigate the effect of the casting solvent on the domain structure. Three different

solvents (toluene, chloroform and ethyl acetate) varying in their polarity are employed to

prepare casting solution. Toluene is a good solvent for PMMA while non-solvent for

P2VP, chloroform is good solvent for both PMMA and P2VP whereas ethyl acetate is

good solvent for P2VP while nonsolvnet for PMMA.

Figure 5-8 shows AFM topographical images of P(VP15-MMA85) copolymer films casted

from toluene, chloroform and ethyl acetate solutions. The surface morphology of the

118

copolymer film casted from toluene was rich in the P2VP phase and poor in PMMA

showing a regular hexagonal pattern. Toluene is a non-polar solvent that has preferred

solvation for PMMA compared to P2VP. Hence, the P2VP phase deposited earlier than

the PMMA phase and formed protrusions over the PMMA domains due to solubility

difference. With the evaporation of toluene PMMA phase finally get deposited onto the

substrate. This phenomenon resulted in a unique morphology of the film in which P2VP

protrudes out of the PMMA domain. Thus, the light region represents the P2VP phase

whereas the dark region represents the PMMA phase. Low surface energy of P2VP

stimulates formation of a continuous phase on the surface while PMMA has to be a

dispersed phase. On the other hand, thin film coated from a chloroform solution produces

a spreaded morphology without preference for any segment. The reason is the good

solubility of both segments in chloroform. On the same lines, if a solvent with higher

polarity is used that has preferential solvation for P2VP compared to PMMA, a different

surface morphology with PMMA phase protrude out on a continuous P2VP phase is

obtained. The spherical morphology of PMMA blocks as protrusions on the P2VP matrix

is observed in this case. PMMA deposited earlier compared to P2VP and formed

protrusions on the P2VP domains. In addition, P2VP has a lower surface free energy

compared to PMMA, hence, P2VP phase has a higher affinity for the air–polymer surface

to obtain a continuous state. Therefore, the polarity and solubility of casting solvent plays

a vital role in the morphology of thin film formed by BCPs.

119

Figure 5-8: Solvent effect on the surface morphology of P(2VP15-MMA85) on Si wafer

5.2.3 Effect of Substrate

As a nest step, the effect of substrate on the morphology of thin films is evaluated by

casting it from toluene. The long- and short-range interactions at air-polymer and

substrate-polymer interfaces resulted in rich interplays and competitions. Block-selective

segregation at the substrate will occur when the wetting component provides the lowest

interfacial tension or exhibits a specific affinity for the substrate, termed as so-called

substrate-induced ordering (Han, Luo, Dai, & Liu, 2008; G. Liu et al., 2009; Tan & Lim,

2004). In this section, three types of substrates are used that have distinct interactions

with PMMA and P2VP segments. Chloroform is selected as casting solvent since it has

no preferential solvation of any of the segment of BCP.

120

Figure 5-9: AFM 3D phase images of P(2VP15-MMA85) block copolymers showing the

polymer-surface and polymer-air interaction of the film cast from chloroform on the

various substartes.

Figure 5-9 presents AFM 3D phase images of the P(VP15-MMA85) copolymer thin films

on mica, silicon, and graphite surfaces, respectively. Mica is hydrophilic and highly polar

ion, Si is hydrophilic and moderately polar whereas graphite is non-polar and

hydrophobic in nature. Apparently, the shape and size of the copolymer thin film coated

on different substrate are not similar. The component surface fraction of the film coated

on the surface is significantly different because the P2VP and PMMA blocks have

different attractions for different substrates. The P(VP15-MMA85) contains a long chain of

PMMA compared to P2VP, therefore, due to high hydrophobic-hydrophobic interaction

on HOPG form a very thin film on the surface. Whereas a slightly thick film is formed on

silicon compared to mica. The reason might be strong interaction of P2VP block with

highly polar mica surface. The mica substrate contains silicon–hydroxyl bonds that

interact with P2VP blocks rendering P2VP blocks distribution over whole mica substrate.

The surface on mica is thicker comparatively observed on HOPG is due to long chain of

PMMA block. Although the silicon substrate has no or little interaction with both PMMA

and P2VP blocks, however, P2VP segments have lower free energy compared to PMMA

segments therefore P2VP segments are enriched at the air–polymer interface to minimize

the air–polymer interfacial free energy. Hence, higher P2VP mole fraction is observed at

the air–polymer interface compared to bulk that resulted in appearance of a lamellar

structure parallel to the surface. Furthermore, the grain height obtained on mica (42.3 nm)

is higher compared to silicon (26.8 nm) due to different surface energies and polymer-

substrate interactions, Figure 5-10.

121

Figure 5-10: Height profile of P(VP15-MMA85) on various substrates (A) HOPG (B) Si

wafer (C) Mica

5.2.4 Thermal Annealing and Surface Morphology

In the following discussion, the effect of thermal annealing on the surface morphology of

the P(2VP-MMA) and P(2VP-MMA)-AuNPs copolymer thin films casted from

chloroform is elaborated. Figure 5-11 shows AFM topographical images for the annealed

films corresponding to those in Figure 5-8. The morphologies of the films were not clear

and showed poorly order prior to thermal annealing. The solubility differences of both

segments and different surface energies might be the reasons of poor ordering of the

polymer chains. Rapid evaporation does not allow enough time to different segments to

position themselves might be another cause of disordered structure. Therefore, surface

structures of P(VP15-MMA85) film annealed at a temperature range of 70 to 230 ⁰C for

about 30 min were studied. An interesting phenomenon was observed for the annealed

specimens with a P2VP fraction. Figure 5-11 shows that the The PMMA and P2VP phase

region were same in untreated sample, however, P2VP region appeared to be greater

while films are annealed at higher temperature. The annealing temperature of 70 ⁰C is

higher than the glass transition temperature of P2VP (50 ⁰C) and was lower than the glass

transition temperature of the PMMA block (100 ⁰C), hence, P2VP segments are more

mobile and resulting domain increased. Furthermore, the annealing of films 110 ⁰C, a

temperature above glass transition temperature of both P2VP and PMMA, higher

immiscibility of both segments is evident. However, the PMMA block and P2VP block

were still in a phase-segregated state during the annealing. Hence, the PMMA segments

122

domains increased again due to increased mobility of PMMA segment beyond its Tg.

Moreover, slight melting of PMMA block started after 110 ⁰C and beyond 160⁰C (i.e. the

melting point of PMMA) dewetting of polymer was observed.

Figure 5-11: Effect of thermal annealing on the morphology of P(2VP15-MMA85)

copolymer film on Si wafer for 30 min. Scale bar on each image is 1µm

To confirm the whole phenomena of thermal annealing of P(2VP15-MMA85) films

morphology, P(2VP-MMA)-AuNPs thin films casted from chloroform were also

annealed vis a vis. Presence of metallic nanoparticles in the P2VP domain resulted in

additional absorption of heat at 70 ⁰C, therefore, P2VP phase extended more compared to

P(2VP15-MMA85). However, at the PMMA segment started to expanding after 110 ⁰C

resulting in increase in the PMMA phase. Finally, the whole morphology was disturb

completely at 160 ⁰C, Figure 5-12.

123

Figure 5-12: Thermal annealed films of P(2VP-MMA)-AuNPs on Si wafer for 30 min

5.3 Conclusion

AFM is the powerful technique for the characterization of self-assemblies of block

copolymers. It is demonstrated that the morphology of P(2VP-MMA) copolymer thin

films can be controlled by varying the total molar mass and individual chain lengths of a

P(2VP-MMA). The factors that can influence the morphology of thin films include total

molar mass of BCP, individual block lengths, solvent used for casting and substrate.

Moreover, gold nanoparticles incorporated with the polymer, completely shielded by

P2VP chains segregate toward the center of the P2VP domain and influenced the

morphology of block copolymer organization by enlarging the polymer domain. Surface

roughness and thickness increased with the increases of molecular weight of polymer.

The morphology on different substrates showed a rough surface for hydrophilic Si wafer

and mica, while flatter for hydrophobic graphite due to different surface interactions of

different substrates. Also, thermal annealing of the polymer casted from chloroform on

the silicon substrate showed that at various temperatures the morphology of the polymer

changes. While at the temperature of 220 ⁰C it became more organized and not further

changes were observed with further rise in temperature.

124

Chapter 6

CONCLUSION

Polymers are large molecules, termed as macromolecules, consisted of small repeated

subunits called monomers. Functionality of the monomer, their arrangement in the

polymer and degree of polymerization define the chemical and physical properties of

polymers as well as the broad range of applications of these polymers in various fields

such as electronics, catalysis, chemical and biochemical sensors, optical devices,

nanosciences and nanotechnology etc.

Nanotechnology deals the matter at the scale of 1-100 nm. At this scale the properties of

matter show different behavior than its bulk material. The optical response of NPs depends

upon size, shape and interparticle distances. For example, gold is an inert metal in normal

condition while at nanoscale it is highly reactive, having good optical response, conduct

electricity and provide a large surface to volume ratio. Among all these advantages the

drawback of these nanoparticles is its stability at ambient conditions, they aggregate and

agglomerate rapidly after formation. Therefore, various stabilizing agents such as various

natural macromolecules such as proteins, flavonoids, liposomes and polysaccharides as

well as synthetic macromolecules such as polymers have been employed for stabilization

of NPs. In this context, we are concerned about synthetic polymers. In literature many

research group used many polymers containing pyrrole, pyridine, thiol or oxygen moiety

are used to stabilize the metallic nanoparticles. Among these homopolymers and block

copolymers of poly(2-vinylpyridine) are widely used because the presence of nitrogen

atoms along with their lone pair of electrons make it suitable for chelating the metallic

nanoparticles and also prevent the nanoparticles from aggregation. Moreover, the

polymer sterically stabilized the nanoparticles so the maximum surface of nanoparticles

are available for further applications. It is seen that the molar mass of P2VP affect the

tendency of P2VP to protect the NPs from aggregation and flocculation. On the other

hand in block copolymers of P2VP, the other block enhanced the properties of P2VP in

reduction of the size of NPs and their protection against aggregation.

125

In this study, we synthesized the gold and silver nanoparticles using P2VP

homopolymers and block copolymers containing P2VP including polystyrene-block-

poly(2-vinylpyridine) and poly(2-vinylpyridine)-block-poly(methyl methacrylate).

Synthesized nanoparticles were characterized by UV-visible spectroscopy, FTIR,

zetasizer, DLS and AFM.

In our first study, we used different molar mass P2VP homopolymers ranging from 1000-

20,000 g/mol to stabilize the AuNPs in one-pot two phase system containing methanol

and water (10:90). It was observed that the small amount of P2VP stabilized the AuNPs

very efficiently as well as the size and stability of AuNPs were greatly controlled at

atomic level through control over the molar mass of P2VP. As the molar mass of P2VP

increases the reducing activity of P2VP increased due to availability of more nitrogen

atoms results in smaller sized AuNPs. It was also observed that P2VP stabilized AuNPs

were stable up to 6 months at ambient temperature and move towards higher stability as

the temperature rises. AuNPs are stable at higher pH values and low electrolyte

concentrations. Moreover, the amount of P2VP also effect on the size and stability of

AuNPs. Drug loading efficiency of these AuNPs stabilized by various molar masses of

P2VP were also established and it was seen that as the size of NPs decreases the drug

encapsulation efficiency of AuNPs increases or in other words, drug encapsulation

efficiency depend upon the molar mass of P2VP.

We were also synthesized the P(S-VP) stabilized AgNPs based nanosensor via two phase

one pot protocol and used these AgNPs for the rapid quantitative determination of one of

the thiocarbamate pesticides. Basically, the NPs have greatly respond towards their

surrounding environment. The presence of any specie that have any kind of secondary

interactions such as hydrogen bonding, van der waals forces, π-π stacking, host-guest

interaction, charge transfer, electrostatic attraction, and antigen-antibody interactions etc.

towards NPs play a vital role in chemosensing. Here, we were screened various kind of

pesticides belongs to different class of pesticides and observed that P(S-VP) stabilized

AgNPs very effectively recognize a thiocarbamate pesticide, named cartap, in real

samples such as tap water, surface runoff water and human blood plasma, also in the

presence of other interfering pesticides and ions at ambient conditions. We characterized

126

and studied the P(S-VP) stabilized AgNPs and its interaction with cartap using UV-

visible spectroscopy, FTIR, zetasizer and AFM. It follows linear correlation with cartap

down to a concentration of 0.06 μgL−1

. We supposed that the optimized P(S-VP)-AgNPs

based quantitative assay would potentially lead to more practical applications because of

its low cost, simple preparation, excellent selectivity, and low detection limit.

Furthermore, we developed a P(2VP3-MMA97)-AuNPs-GCE based electrochemical

sensor for rapid quantitative assay of nicotine. In starting we selected the three different

polymers having different individual blocks and molar masses such as P(2VP3-MMA97)

(23300 g/mol), P(2VP18-MMA85) (28300 g/mol) and P(2VP10-MMA90) (40400 g/mol)

and synthesized the AuNPs. But it was observed that AuNPs synthesized with P(2VP3-

MMA97) gives far better performance as electrochemical sensor for nicotine when coated

on GCE electrode than P(2VP18-MMA85) and P(2VP10-MMA90) in organic media.

Therefore, for further studies we used the P(2VP3-MMA97)-AuNPs for nicotine detection.

A well-developed voltammetric peak appeared at +0.66 V (versus Ag/Ag+), in

acetonitrile for determination of nicotine in the concentration range of 0.1 – 0.4 mM with

a detection limit of 0.16 mM. The P(2VP3-MMA97)-AuNPs-GCE is more sensitive

towards nicotine, the electrochemical response obtained by P(2VP3-MMA97)-AuNPs-

GCE is enhanced by an enhancement factor of ~2 compared to bare GCE. Moreover, the

P(2VP3-MMA97)-AuNPs were smaller in size and the stability of P(2VP3-MMA97)-

AuNPs against electrolyte, pH and temperature are higher than P(2VP18-MMA85) and

P(2VP10-MMA90). Stability and homogeneous nature of P(2VP3-MMA97)-AuNPs was

confirmed by UV-Vis, FTIR, AFM, and zetasizer.

We studied the morphology of P(2VP-MMA) using AFM which is the powerful

technique for the surface characterization of thin films of self-assembled block

copolymers. These block copolymer (BCP) films offer imaginable self-organized

patterned morphologies of molecular dimensions in a highly ordered way that is desirable

when working towards miniaturization of electronic and optical devices. Depending on

the length, connectivity, and mutual interactions of the different blocks, the

microdomains can form spherical, lamellar, cylindrical, gyroid, or more complex shapes,

which exhibit regular periodic order with typical repeat distances in the range between

127

10-100 nm. Various factors affecting the surface morphology of thin films of polymers,

such as the molecular weight and composition of block copolymer, casting solvent,

annealing temperature, film thickness, interfacial interactions, solvent evaporation,

substrate pattern, and electric fields. By various experimentations, we have demonstrated

that the morphology of P(2VP-MMA) copolymer thin films can be controlled simply by

varying the chain length of a P(2VP-MMA) chain. It was observed that both P2VP and

PMMA individual block, total molecular weight of block, solvent used for casting and

substrate play a vital role to decide the final morphology of block copolymer thin film.

Moreover, gold nanoparticles incorporated with the polymer, completely shielded by

P2VP chains segregate toward the center of the P2VP domain and influenced the

morphology of block copolymer organization by enlarging the polymer domain. Surface

roughness and thickness are increased with the increase of molecular weight of polymer.

The morphology on different substrates showed a rough surface for hydrophilic Si wafer

and mica, while flatter for hydrophobic graphite due to different surface interactions of

different substrates.

128

References

Abraham, S., Kim, I., & Batt, C. A. (2007). A Facile Preparative Method for

Aggregation-Free Gold Nanoparticles Using Poly(styrene-block-cysteine).

Angew. Chem. Int. Ed., 46(30), 5720-5723. doi: 10.1002/anie.200701060

Ackerson, C. J., Jadzinsky, P. D., & Kornberg, R. D. (2005). Thiolate ligands for

synthesis of water-soluble gold clusters. JACS, 127(18), 6550-6551.

Aharonov, Y., & Bohm, D. (1959). Significance of electromagnetic potentials in the

quantum theory. Phys. Rev., 115(3), 485.

Ahmed, F., Khalid, S., Shah, K., & Shah, M. R. (2016). A Highly Sensitive and Selective

Supramolecular Fluorescent Chemosensor for Dichromate Ion Detection and

Application to Real Samples. J. Chem. Soc. Pak., 38(1), 171-176.

Alberg, A. J. (2008). Cigarette smoking: health effects and control strategies. Drugs

today, 44(12), 895-904.

Alexandridis, P. (1996). Amphiphilic copolymers and their applications. Curr. Opin.

Colloid Interface Sci., 1(4), 490-501. doi: http://dx.doi.org/10.1016/S1359-0294

(96)80118-X

Anwar, A., Shah, M. R., Muhammad, S. P., Afridi, S., & Ali, K. (2016). Thio-pyridinium

capped silver nanoparticle based supramolecular recognition of Cu (I) in real

samples and T-lymphocytes. New J. Chem., 40(7), 6480-6486.

Aragay, G., Pino, F., & Merkoçi, A. (2012). Nanomaterials for sensing and destroying

pesticides. Chem. Rev., 112(10), 5317-5338.

Armitage, A., & Hall, G. (1967). Further evidence relating to the mode of action of

nicotine in the central nervous system. Nature, 214(5092), 977-979.

129

Ateeq, M., Shah, M. R., ul Ain, N., Bano, S., Anis, I., Faizi, S., Naz, S. S. (2015). Green

synthesis and molecular recognition ability of patuletin coated gold nanoparticles.

Biosens. Bioelectron., 63, 499-505.

Atwood, J. W. S. a. J. L. (2009). Supramolecular Chemistry (2nd ed.). United kingdom:

John Wiley & Sons, Ltd.

Aurélien, S., Arthur, G., Alexandre, P., Jérémie, B., Hélène, Y.-L., Jean-Louis, B., . . .

Thomas, M. (2014). Single step synthesis and organization of gold colloids

assisted by copolymer templates. Nanotechnol., 25(22), 225603.

Badawy, A. M. E., Luxton, T. P., Silva, R. G., Scheckel, K. G., Suidan, M. T., &

Tolaymat, T. M. (2010a). Impact of Environmental Conditions (pH, Ionic

Strength, and Electrolyte Type) on the Surface Charge and Aggregation of Silver

Nanoparticles Suspensions. Environ. Sci. Technol., 44(4), 1260-1266. doi: 10.102

1/es902240k

Badawy, A. M. E., Luxton, T. P., Silva, R. G., Scheckel, K. G., Suidan, M. T., &

Tolaymat, T. M. (2010b). Impact of Environmental Conditions (pH, Ionic

Strength, and Electrolyte Type) on the Surface Charge and Aggregation of Silver

Nanoparticles Suspensions. Environ. Sci. Technol., 44(4), 1260-1266. doi:

10.1021/es902240k

Bae, C. H., Nam, S. H., & Park, S. M. (2002). Formation of silver nanoparticles by laser

ablation of a silver target in NaCl solution. Appl. Surf. Sci. , 197, 628-634.

Balazs, A. C., Emrick, T., & Russell, T. P. (2006). Nanoparticle Polymer Composites:

Where Two Small Worlds Meet. Science, 314(5802), 1107-1110. doi: 10.1126/

science.1130557

Bawendi, M. G., Sundar, V. C., & Mikulec, F. V. (2007). Biological applications of quantum

dots: Google Patents.

130

Bhargava, R., Wang, S.-Q., & Koenig, J. L. (2003). FTIR Microspectroscopy of

Polymeric Systems Liquid Chromatography / FTIR Microspectroscopy /

Microwave Assisted Synthesis (Vol. 163, pp. 137-191): Springer Berlin

Heidelberg.

Bhatia, S. (2016). Nanoparticles Types, Classification, Characterization, Fabrication

Methods and Drug Delivery Applications Natural Polymer Drug Delivery

Systems (pp. 33-93): Springer.

Bhumkar, D. R., Joshi, H. M., Sastry, M., & Pokharkar, V. B. (2007). Chitosan reduced

gold nanoparticles as novel carriers for transmucosal delivery of insulin. Pharm.

Res., 24(8), 1415-1426.

Bindhu, M., & Umadevi, M. Green Synthesized Gold Nanoparticles as a Probe for the

Detection of Fe3+ Ions in Water. J. Cluster Sci., 1-10.

Blanchard, C. R. (1996). Atomic Force Microscopy. Chem. Educ., 1(5), 1-8. doi: 10.10

07/s00897960059a

Boal, A. K., Ilhan, F., DeRouchey, J. E., & Thurn-Albrecht, T. (2000). Self-assembly of

nanoparticles into structured spherical and network aggregates. Nature,

404(6779), 746.

Bockstaller, M. R., Mickiewicz, R. A., & Thomas, E. L. (2005). Block copolymer

nanocomposites: perspectives for tailored functional materials. Adv. Mater., 17

(11), 1331-1349.

Bockstaller, M. R., & Thomas, E. L. (2003). Optical properties of polymer-based

photonic nanocomposite materials. J. Phys. Chem. B, 107(37), 10017-10024.

Boisselier, E., & Astruc, D. (2009). Gold nanoparticles in nanomedicine: preparations,

imaging, diagnostics, therapies and toxicity. Chem. Soc. Rev., 38(6), 1759-1782.

Bouzigues, C., Gacoin, T., & Alexandrou, A. (2011). Biological applications of rare-earth

based nanoparticles. ACS Nano, 5(11), 8488-8505.

131

Bronstein, L. M., Sidorov, S. N., Valetsky, P. M., Hartmann, J., Cölfen, H., & Antonietti,

M. (1999). Induced Micellization by Interaction of Poly(2-vinylpyridine)-block-

poly(ethylene oxide) with Metal Compounds. Micelle Characteristics and Metal

Nanoparticle Formation. Langmuir, 15(19), 6256-6262. doi: 10.1021/la990146f

Bruns, N., & Tiller, J. C. (2005). Amphiphilic network as nanoreactor for enzymes in

organic solvents. Nano Lett., 5(1), 45-48.

Brust, M., Walker, M., Bethell, D., Schiffrin, D. J., & Whyman, R. (1994). Synthesis of

thiol-derivatised gold nanoparticles in a two-phase liquid–liquid system. J. Chem.

Soc., Chem. Commun. (7), 801-802.

Cameron, J. M., Howell, D. N., White, J. R., Andrenyak, D. M., Layton, M. E., & Roll, J.

M. (2014). Variable and potentially fatal amounts of nicotine in e-cigarette

nicotine solutions. Tob. control, 23(1), 77-78.

Campoy-Quiles, M., Ferenczi, T., Agostinelli, T., Etchegoin, P. G., Kim, Y.,

Anthopoulos, T. D., Nelson, J. (2008). Morphology evolution via self-

organization and lateral and vertical diffusion in polymer: fullerene solar cell

blends. Nat. Mater., 7(2), 158.

Cao, Y., Zhang, A., Ma, Q., Liu, N., & Yang, P. (2013). Application of hybrid SiO2‐

coated CdTe nanocrystals for sensitive sensing of Cu2+ and Ag+ ions. J. Lumin.,

28(3), 287-293.

Carotenuto, G. (2001). Synthesis and characterization of poly(N-vinylpyrrolidone) filled

by monodispersed silver clusters with controlled size. Appl. Organomet. Chem.,

15(5), 344-351. doi: 10.1002/aoc.165

Carotenuto, G., Pepe, G. P., & Nicolais, L. (2000). Preparation and characterization of

nano-sized Ag/PVP composites for optical applications. Eur. Phys. J. B, 16(1),

11-17. doi: 10.1007/s100510070243

132

Caruso, F., Caruso, R. A., & Möhwald, H. (1998). Nanoengineering of inorganic and

hybrid hollow spheres by colloidal templating. Science, 282(5391), 1111-1114.

Casida, J. E., & Quistad, G. B. (1998). Golden age of insecticide research: past, present,

or future? Ann. Rev. Entomol., 43(1), 1-16.

Chah, S., Hammond, M. R., & Zare, R. N. (2005). Gold Nanoparticles as a Colorimetric

Sensor for Protein Conformational Changes. Chem. Biol., 12(3), 323-328. doi:

http://dx.doi.org/10.1016/j.chembiol.2005.01.013

charles E. Carraher, J. (2012). Introduction to polymer chemistry (Third ed.). New York.

Cheng, J. Y., Ross, C. A., Chan, V. Z. H., Thomas, E. L., Lammertink, R. G. H., &

Vancso, G. J. (2001). Formation of a cobalt magnetic dot array via block

copolymer lithography. Adv. Mater., 13(15), 1174-1178.

Chernousova, S., & Epple, M. (2013). Silver as antibacterial agent: ion, nanoparticle, and

metal. Angew. Chem. Int. Ed., 52(6), 1636-1653.

Chiu, J. J., Kim, B. J., Kramer, E. J., & Pine, D. J. (2005). Control of Nanoparticle

Location in Block Copolymers. J. Am. Chem. Soc., 127(14), 5036-5037. doi:

10.1021/ja050376i

Compton, R. G., & Banks, C. E. (2007). Understanding voltammetry: World Scientific.

Corthey, G. n., Rubert, A. A., Picone, A. L., Casillas, G., Giovanetti, L. J -

-

thiolate-protected palladium nanoparticles. J. Phys. Chem. C, 116(17), 9830-

9837.

Crooks, R. M., Zhao, M., Sun, L., Chechik, V., & Yeung, L. K. (2001). Dendrimer-

Encapsulated Metal Nanoparticles: Synthesis, Characterization, and Applications

to Catalysis. Acc. Chem. Res., 34(3), 181-190. doi: 10.1021/ar00011 0a

133

Cui, L., Ding, Y., Li, X., Wang, Z., & Han, Y. (2006). Solvent and polymer concentration

effects on the surface morphology evolution of immiscible polystyrene/poly

(methyl methacrylate) blends. Thin Solid Films, 515(4), 2038-2048.

Dadwal, M., Solan, D., & Pradesh, H. (2014). Polymeric nanoparticles as promising

novel carriers for drug delivery: An overview. J. Adv. Pharm. Educ. Res., 4(1).

Daniel, M.-C., & Astruc, D. (2004). Gold nanoparticles: assembly, supramolecular

chemistry, quantum-size-related properties, and applications toward biology,

catalysis, and nanotechnology. Chem. Rev., 104(1), 293-346.

Darling, S. B. (2007). Directing the self-assembly of block copolymers. Progress in

Polymer Science, 32(10), 1152-1204. doi:http://dx.doi.org/10.1016/j.progpoly

msci. 2007. 05. 004

Davis, R. A. (1986). The Determination of Nicotine and Cotinine in Plasma. J.

Chromatogr. Sci., 24(4), 134-141. doi: 10.1093/chromsci/24.4.134

Deraedt, C., Salmon, L., Gatard, S., Ciganda, R., Hernandez, R., Ruiz, J., & Astruc, D.

(2014). Sodium borohydride stabilizes very active gold nanoparticle catalysts.

Chem. Commun., 50(91), 14194-14196.

Drbohlavova, J., Adam, V., Kizek, R., & Hubalek, J. (2009). Quantum dots—

characterization, preparation and usage in biological systems. Int. J. Mol. Sci.,

10(2), 656-673.

El-Nour, K. M. A., Eftaiha, A. a., Al-Warthan, A., & Ammar, R. A. (2010). Synthesis

and applications of silver nanoparticles. Arabian J. Chem., 3(3), 135-140.

Eldefrawi, A. T., Bakry, N. M., Eldefrawi, M. E., Tsai, M.-C., & Albuquerque, E. X.

(1980). Nereistoxin interaction with the acetylcholine receptor-ionic channel

complex. Mol. Pharmacol., 17(2), 172-179.

Electroanalytical Methods - Guide to Experiments and Applications. (2010). (F. E.

Scholz Ed. Vol. 1). Berlin Heidelberg: Springer.

134

Everett, W. R., & Rechnitz, G. A. (1998). Mediated bioelectrocatalytic determination of

organophosphorus pesticides with a tyrosinase-based oxygen biosensor. Anal.

Chem., 70(4), 807-810.

Fahmi, A., Pietsch, T., Mendoza, C., & Cheval, N. (2009). Functional hybrid materials.

Mater. Today, 12(5), 44-50. doi: http://dx.doi.org/10.1016/S1369-7021(09)70159-

2

Fang, R., Jing, H., Chai, Z., Zhao, G., Stoll, S., Ren, F., . . . Leng, X. (2011). Study of the

physicochemical properties of the BSA: Flavonoid nanoparticle. Eur. Food Res.

Technol., 233(2), 275-283. doi: 10.1007/s00217-011-1522-9

Fisher, D. H., Xie, Y., & Loring, R. (1993). Analysis of nereistoxin using HPLC and

electrochemical detection. Anal. Lett., 26(6), 1051-1063.

Förster, H. (2004). UV/VIS Spectroscopy. In H. G. Karge & J. Weitkamp (Eds.),

Characterization I: (pp. 337-426). Berlin, Heidelberg: Springer Berlin

Heidelberg.

Frederix, F., Friedt, J.-M., Choi, K.-H., Laureyn, W., Campitelli, A., Mondelaers, D., . . .

Borghs, G. (2003). Biosensing based on light absorption of nanoscaled gold and

silver particles. Anal. Chem., 75(24), 6894-6900.

Frens, G. (1973). Controlled nucleation for the regulation of the particle size in

monodisperse gold suspensions. Nature, 241(105), 20-22.

Fu, Q., & Sun, W. (2001). Mie theory for light scattering by a spherical particle in an

absorbing medium. Applied Optics, 40(9), 1354-1361.

Galatsis, K., Wang, K. L., Ozkan, M., Ozkan, C. S., Huang, Y., Chang, J. P., . . . Botros,

Y. (2010). Patterning and templating for nanoelectronics. Adv. Mater., 22(6), 769-

778.

Gandubert, V. J., & Lennox, R. B. (2005a). Assessment of 4-(dimethylamino) pyridine as

a capping agent for gold nanoparticles. Langmuir, 21(14), 6532-6539.

http://dx.doi.org/10.1016/S1369-7021(09)70159-2

http://dx.doi.org/10.1016/S1369-7021(09)70159-2

135

Gandubert, V. J., & Lennox, R. B. (2005b). Assessment of 4-(dimethylamino) pyridine as

a capping agent for gold nanoparticles. Langmuir, 21(14), 6532-6539.

Gandubert, V. J., & Lennox, R. B. (2005c). Assessment of 4-(Dimethylamino)pyridine as

a Capping Agent for Gold Nanoparticles. Langmuir, 21(14), 6532-6539. doi:

10.1021/la050195u

Gindy, M. E., Panagiotopoulos, A. Z., & Prud'homme, R. K. (2008). Composite block

copolymer stabilized nanoparticles: simultaneous encapsulation of organic actives

and inorganic nanostructures. Langmuir, 24(1), 83-90.

Gittins, D. I., & Caruso, F. (2001). Spontaneous phase transfer of nanoparticulate metals

from organic to aqueous media. Angew. Chem. Int. Ed., 40(16), 3001-3004.

Gittins, D. I., & Caruso, F. (2001). Spontaneous phase transfer of nanoparticulate metals

from organic to aqueous media. Angew. Chem. Int. Ed., 40(16), 3001-3004.

Goldburg, W. I. (1999). Dynamic light scattering. Am. J. Phys., 67(12), 1152-1160. doi:

doi:http://dx.doi.org/10.1119/1.19101

Goniewicz, M. L., Kuma, T., Gawron, M., Knysak, J., & Kosmider, L. (2013). Nicotine

Levels in Electronic Cigarettes. Nicotine Tob. Res., 15(1), 158-166. doi:

10.1093/ntr/nts103

Gonzalez, J. M., Foley, M. W., Bieber, N. M., Bourdelle, P. A., & Niedbala, R. S. (2011).

Development of an ultrasensitive immunochromatography test to detect nicotine

metabolites in oral fluids. Anal. Bioanal. Chem., 400(10), 3655-3664. doi:

10.1007/s00216-011-5051-y

Grzelczak, M., Vermant, J., Furst, E. M., & Liz-Marzán, L. M. (2010). Directed self-

assembly of nanoparticles. ACS nano, 4(7), 3591-3605.

Guo, J., Liu, X., Gao, H., Bie, J., Zhang, Y., Liu, B., & Sun, C. (2014). Highly sensitive

turn-on fluorescent detection of cartap via a nonconjugated gold nanoparticle–

quantum dot pair mediated by inner filter effect. RSC Adv., 4(52), 27228-27235.

http://dx.doi.org/10.1119/1.19101

136

Han, X., Luo, C., Dai, Y., & Liu, H. (2008). Effect of Polymer-Substrate Interactions on

the Surface Morphology of Polymer Blend Thin Films. J. Macromol. Sci. Part B

Phys., 47(6), 1050-1061. doi: 10.1080/00222340802266322

Harsha, T., Abhilash, K., & Hansdak, S. (2013). Cartap Hydrochloride Poisoning: A

Clinical Experience with N-Acetyl Cysteine Therapy. J. Med. Sci. Res., 4(1), 30.

Haruta, M. (1997). Size-and support-dependency in the catalysis of gold. Catal. Today,

36(1), 153-166.

Haruta, M., & Daté, M. (2001). Advances in the catalysis of Au nanoparticles. Appl.

Catal., A: General, 222(1-2), 427-437.

Haruta, M., Kobayashi, T., Sano, H., & Yamada, N. (1987). Novel gold catalysts for the

oxidation of carbon monoxide at a temperature far below 0 C. Chem. Lett., 16(2),

405-408.

Haruta, M., Tsubota, S., Kobayashi, T., Kageyama, H., Genet, M. J., & Delmon, B.

(1993). Low-temperature oxidation of CO over gold supported on TiO2, α-

Fe2O3, and Co3O4. J. Catal., 144(1), 175-192.

Haruta, M., Yamada, N., Kobayashi, T., & Iijima, S. (1989). Gold catalysts prepared by

coprecipitation for low-temperature oxidation of hydrogen and of carbon

monoxide. J. Catal., 115(2), 301-309.

Haryono, A., & Binder, W. H. (2006). Controlled Arrangement of Nanoparticle Arrays in

Block‐Copolymer Domains. Small, 2(5), 600-611.

Hattori, Y., Mukasa, S., Toyota, H., Inoue, T., & Nomura, S. (2011). Synthesis of zinc

and zinc oxide nanoparticles from zinc electrode using plasma in liquid. Mater.

Lett., 65(2), 188-190.

Heinze, J. (1984). Cyclic voltammetry—―electrochemical spectroscopy‖. New analytical

methods (25). Angew. Chem. Int. Ed., 23(11), 831-847.

137

Henry, C. R. (2000). Catalytic activity of supported nanometer-sized metal clusters. Appl.

Surf. Sci., 164(1), 252-259.

Highton, L., Kadara, R. O., Jenkinson, N., Logan Riehl, B., & Banks, C. E. (2009).

Metallic Free Carbon Nanotube Cluster Modified Screen Printed Electrodes for

the Sensing of Nicotine in Artificial Saliva. Electroanalysis, 21(21), 2387-2389.

doi: 10.1002/elan.200904683

Hill, A. V. (1909). The mode of action of nicotine and curari, determined by the form of

the contraction curve and the method of temperature coefficients. J. Physiol.,

39(5), 361-373.

Huh, J., Ginzburg, V. V., & Balazs, A. C. (2000). Thermodynamic Behavior of

Particle/Diblock Copolymer Mixtures: Simulation and Theory. Macromolecules,

33(21), 8085-8096. doi: 10.1021/ma000708y

Ikkala, O., & ten Brinke, G. (2004). Hierarchical self-assembly in polymeric complexes:

towards functional materials. Chem. Commun.(19), 2131-2137.

Jadzinsky, P. D., Calero, G., Ackerson, C. J., Bushnell, D. A., & Kornberg, R. D. (2007).

Structure of a thiol monolayer-protected gold nanoparticle at 1.1 Å resolution.

Science, 318(5849), 430-433.

Jang, S. G., Khan, A., Hawker, C. J., & Kramer, E. J. (2012). Morphology Evolution of

PS-b-P2VP Diblock Copolymers via Supramolecular Assembly of Hydroxylated

Gold Nanoparticles. Macromolecules, 45(3), 1553-1561. doi: 10.1021/ma20239

1k

Jaramillo, T. F., Baeck, S.-H., Cuenya, B. R., & McFarland, E. W. (2003). Catalytic

Activity of Supported Au Nanoparticles Deposited from Block Copolymer

Micelles. J. Am. Chem. Soc., 125(24), 7148-7149. doi: 10.1021/ja029800v

Jiang, J., Oberdörster, G., & Biswas, P. (2009a). Characterization of size, surface charge,

and agglomeration state of nanoparticle dispersions for toxicological studies. J.

Nanopart. Res., 11(1), 77-89.

138

Jiang, J., Oberdörster, G., & Biswas, P. (2009b). Characterization of size, surface charge,

and agglomeration state of nanoparticle dispersions for toxicological studies. J.

Nanopart. Res., 11(1), 77-89.

Jin, J., Iyoda, T., Cao, C., Song, Y., Jiang, L., Li, T. J., & Zhu, D. B. (2001). Self‐

Assembly of Uniform Spherical Aggregates of Magnetic Nanoparticles through

π–π Interactions. Angew. Chem. Int. Ed., 40(11), 2135-2138.

Jin, T., & He, Y. (2011). Antibacterial activities of magnesium oxide (MgO)

nanoparticles against foodborne pathogens. J. Nanopart. Res., 13(12), 6877-6885.

Jing, X., Du, L.-m., Wu, H., Wu, W.-y., & Chang, Y.-x. (2012). Determination of

Pesticide Residue Cartap Using a Sensitive Fluorescent Probe. Journal of

Integrative Agriculture, 11(11), 1861-1870. doi: https://doi.org/10.1016/S2095-

3119(12)60191-9

Jing, Y., Yuan, X., Yuan, Q., He, K., Liu, Y., Lu, P., Li, G. (2016). Determination of

nicotine in tobacco products based on mussel-inspired reduced graphene oxide-

supported gold nanoparticles. Sci. Rep., 6, 29230. doi: 10.1038/srep29230

Jo, J., Kim, S. S., Na, S. I., Yu, B. K., & Kim, D. Y. (2009). Time‐Dependent

Morphology Evolution by Annealing Processes on Polymer: Fullerene Blend

Solar Cells. Adv. Funct. Mater., 19(6), 866-874.

Jores, K., Mehnert, W., Drechsler, M., Bunjes, H., Johann, C., & Mäder, K. (2004).

Investigations on the structure of solid lipid nanoparticles (SLN) and oil-loaded

solid lipid nanoparticles by photon correlation spectroscopy, field-flow

fractionation and transmission electron microscopy. J. Controlled Release 95(2),

217-227.

Ju-Nam, Y., & Lead, J. R. (2008). Manufactured nanoparticles: an overview of their

chemistry, interactions and potential environmental implications. Sci. Total

Environ., 400(1), 396-414.

139

Judefeind, A., & de Villiers, M. M. (2009). Drug loading into and in vitro release from

nanosized drug delivery systems Nanotechnology in drug delivery (pp. 129-162):

Springer.

Kang, Y., & Taton, T. A. (2005). Controlling Shell Thickness in Core−Shell Gold

Nanoparticles via Surface-Templated Adsorption of Block Copolymer

Surfactants. Macromolecules, 38(14), 6115-6121. doi: 10.1021/ma050400c

Kao, J., Thorkelsson, K., Bai, P., Rancatore, B. J., & Xu, T. (2013). Toward functional

nanocomposites: taking the best of nanoparticles, polymers, and small molecules.

Chem. Soc. Rev., 42(7), 2654-2678.

Khandekar, S. V., Kulkarni, M., & Devarajan, P. V. (2014). Polyaspartic acid

functionalized gold nanoparticles for tumor targeted doxorubicin delivery. J.

Biomed. Nanotechnol., 10(1), 143-153.

Kim, B. J., Bang, J., Hawker, C. J., Chiu, J. J., Pine, D. J., Jang, S. G., Kramer, E. J.

(2007). Creating Surfactant Nanoparticles for Block Copolymer Composites

through Surface Chemistry. Langmuir, 23(25), 12693-12703. doi: 10.1021/la7019

06n

Kim, B. J., Bang, J., Hawker, C. J., & Kramer, E. J. (2006). Effect of Areal Chain

Density on the Location of Polymer-Modified Gold Nanoparticles in a Block

Copolymer Template. Macromolecules, 39(12), 4108-4114. doi: 10.1021/ma060

308w

Kim, B. J., Fredrickson, G. H., & Kramer, E. J. (2008). Effect of Polymer Ligand

Molecular Weight on Polymer-Coated Nanoparticle Location in Block

Copolymers. Macromolecules, 41(2), 436-447. doi: 10.1021/ma701931z

Kim, H.-C., Park, S.-M., & Hinsberg, W. D. (2009). Block copolymer based

nanostructures: materials, processes, and applications to electronics. Chem. Rev.,

110(1), 146-177.

140

Kim, J. S., Kuk, E., Yu, K. N., Kim, J.-H., Park, S. J., Lee, H. J., Hwang, C.-Y. (2007).

Antimicrobial effects of silver nanoparticles. Nanomed. Nanotechnol. Biol. Med.,

3(1), 95-101.

Kim, Y., Jung, J., Oh, S., & Choi, K. (2008). Aquatic toxicity of cartap and cypermethrin

to different life stages of Daphnia magna and Oryzias latipes. J. Environ. Sci.

Health., 43(1), 56-64.

Kissinger, P. T., & Heineman, W. R. (1983). Cyclic voltammetry. J. Chem. Educ, 60(9),

702.

Kong, W., Li, B., Jin, Q., Ding, D., & Shi, A.-C. (2010). Complex Micelles from Self-

Assembly of ABA Triblock Copolymers in B-Selective Solvents. Langmuir,

26(6), 4226-4232. doi: 10.1021/la903292f

Kreuter, J. (1983). Physicochemical characterization of polyacrylic nanoparticles. Int. J.

Pharm., 14(1), 43-58.

Kumar, A. P., Amalnath, D., & Dutta, T. (2011). Cartap poisoning: A rare case report.

Indian journal of critical care medicine: peer-reviewed, official publication of

Indian Society of Critical Care Medicine, 15(4), 233.

Kunz, M. S., Shull, K. R., & Kellock, A. J. (1993). Colloidal gold dispersions in

polymeric matrices. J. Colloid Interface Sci., 156(1), 240-249.

Kurisaki, E., Kato, N., Ishida, T., Matsumoto, A., Shinohara, K., & Hiraiwa, K. (2010).

Fatal human poisoning with PadanTM: a cartap-containing pesticide. J. Toxicol.,

48(2), 153-155.

Kwon, G. S., & Kataoka, K. (2012). Block copolymer micelles as long-circulating drug

vehicles. Adv. Drug Delivery Rev., 64, 237-245.

Lambe, J., & Jaklevic, R. (1968). Molecular vibration spectra by inelastic electron

tunneling. Phys. Rev., 165(3), 821.

141

Laviron, E. (1974). Adsorption, autoinhibition and autocatalysis in polarography and in

linear potential sweep voltammetry. J. Electroanal. Chem. Interfacial

Electrochem., 52(3), 355-393.

Lekesiz, T. O., Kayran, C., & Hacaloglu, J. (2015). Preparation and thermal

characterization of poly(2-vinylpyridine) copolymers coordinated to Cr

nanoparticles. Polym. Adv. Technol., 26(6), 555-560. doi: 10.1002/pat.3484

Lévy, R., Thanh, N. T. K., Doty, R. C., Hussain, I., Nichols, R. J., Schiffrin, D. J., Fernig,

D. G. (2004). Rational and Combinatorial Design of Peptide Capping Ligands for

Gold Nanoparticles. JACS, 126(32), 10076-10084. doi: 10.1021/ja0487269

Li, G., Shrotriya, V., Huang, J., Yao, Y., Moriarty, T., Emery, K., & Yang, Y. (2005).

High-efficiency solution processable polymer photovoltaic cells by self-

organization of polymer blends. Nat. Mater., 4(11), 864.

Li, G., Shrotriya, V., Yao, Y., & Yang, Y. (2005). Investigation of annealing effects and

film thickness dependence of polymer solar cells based on poly (3-hexylthiophene).

J. Appl. Phys., 98(4), 043704.

Li, Z., & Zhang, J. (2006). An efficient theoretical study on host–guest interactions of a

fluoride chemosensor with F−, Cl− and Br−. Chem. Phys., 331(1), 159-163.

Liao, J.-W., Kang, J.-J., Jeng, C.-R., Chang, S.-K., Kuo, M.-J., Wang, S.-C., . . . Pang, V.

F. (2006). Cartap-induced cytotoxicity in mouse C 2 C 12 myoblast cell line and

the roles of calcium ion and oxidative stress on the toxic effects. Toxicology,

219(1), 73-84.

Liao, J.-W., Pang, V. F., Jeng, C.-R., Chang, S.-K., Hwang, J.-S., & Wang, S.-C. (2003).

Susceptibility to cartap-induced lethal effect and diaphragmatic injury via ocular

exposure in rabbits. Toxicology, 192(2), 139-148.

Liu, G., Ji, S., Stuen, K. O., Craig, G. S. W., Nealey, P. F., & Himpsel, F. J. (2009).

Modification of a polystyrene brush layer by insertion of poly (methyl

methacrylate) molecules. J. Vac. Sci. Technol., B, 27(6), 3038-3042.

142

Liu, J., Mendoza, S., Román, E., Lynn, M. J., Xu, R., & Kaifer, A. E. (1999).

Cyclodextrin-Modified Gold Nanospheres. Host− Guest Interactions at Work to

Control Colloidal Properties. J. Am. Chem. Soc., 121(17), 4304-4305.

Liu, W., Zhang, D., Tang, Y., Wang, Y., Yan, F., Li, Z., Zhou, H. S. (2012). Highly

sensitive and selective colorimetric detection of cartap residue in agricultural

products. Talanta, 101, 382-387.

Liu, W., Zhang, D., Zhu, W., Zhang, S., Wang, Y., Yu, S., Wang, J. (2015). Colorimetric

and visual determination of total nereistoxin-related insecticides by exploiting a

nereistoxin-driven aggregation of gold nanoparticles. Microchim. Acta, 182(1),

401-408. doi: 10.1007/s00604-014-1347-x

Lo, T. W. B., Aldous, L., & Compton, R. G. (2012). The use of nano-carbon as an

alternative to multi-walled carbon nanotubes in modified electrodes for adsorptive

stripping voltammetry. Sens. Actuators, B, 162(1), 361-368. doi: https://doi.org/1

0.1016/j.snb.2011.12.104

Luo, C., Zhang, Y., Zeng, X., Zeng, Y., & Wang, Y. (2005). The role of poly(ethylene

glycol) in the formation of silver nanoparticles. J. Colloid Interface Sci., 288(2),

444-448. doi: http://dx.doi.org/10.1016/j.jcis.2005.03.005

Lutz, J.-F. (2008). Polymerization of oligo(ethylene glycol) (meth)acrylates: Toward new

generations of smart biocompatible materials. J. Polym. Sci. Part A: Polym.

Chem., 46(11), 3459-3470. doi: 10.1002/pola.22706

Ma, W., Yang, C., Gong, X., Lee, K., & Heeger, A. J. (2005). Thermally stable, efficient

polymer solar cells with nanoscale control of the interpenetrating network

morphology. Adv. Funct. Mater., 15(10), 1617-1622.

Macheroux, P. (1999). UV-Visible Spectroscopy as a Tool to Study Flavoproteins. In S.

K. Chapman & G. A. Reid (Eds.), Flavoprotein Protocols (pp. 1-7). Totowa, NJ:

Humana Press.

http://dx.doi.org/10.1016/j.jcis.2005.03.005

143

Magenheim, B., Levy, M., & Benita, S. (1993). A new in vitro technique for the

evaluation of drug release profile from colloidal carriers-ultrafiltration technique

at low pressure. Int. J. Pharm. , 94(1-3), 115-123.

Magonov, S. N., Bar, G., Cantow, H. J., Bauer, H. D., Müller, I., & Schwoerer, M.

(1991). Atomic force microscopy on polymers and polymer related compounds.

Polym. Bull., 26(2), 223-230. doi: 10.1007/BF00297531

Mahoney, G. N., & Al-Delaimy, W. (2001). Measurement of nicotine in hair by reversed-

phase high-performance liquid chromatography with electrochemical detection. J.

Chromatogr. B, 753(2), 179-187. doi: https://doi.org/10.1016/S0378-4347(00)0

0540-5

Mamián-López, M. B., & Poppi, R. J. (2013). Standard addition method applied to the

urinary quantification of nicotine in the presence of cotinine and anabasine using

surface enhanced Raman spectroscopy and multivariate curve resolution. Anal.

Chim. Acta, 760(Supplement C), 53-59. doi: https://doi.org/10.1016/j.aca.2012.1

1.023

Matysik, F.-M. (1999). Application of non-aqueous capillary electrophoresis with

electrochemical detection to the determination of nicotine in tobacco. J.

Chromatogr. A, 853(1), 27-34. doi: https://doi.org/10.1016/S0021-9673(99)005

12-9

McManus, K. T., deBethizy, J. D., Garteiz, D. A., Kyerematen, G. A., & Vesell, E. S.

(1990). A new quantitative thermospray LC-MS method for nicotine and its

metabolites in biological fluids. J. Chromatogr. Sci., 28(10), 510-516.

Mei, L., Somesfalean, G., & Svanberg, S. (2014). Pathlength Determination for Gas in

Scattering Media Absorption Spectroscopy. Sensors, 14(3), 3871.

Mei, Y., Lu, Y., Polzer, F., Ballauff, M., & Drechsler, M. (2007). Catalytic Activity of

Palladium Nanoparticles Encapsulated in Spherical Polyelectrolyte Brushes and

Core−Shell Microgels. Chem. Mater., 19(5), 1062-1069. doi: 10.1021/cm062554s

144

Meulenkamp, E. A. (1998). Synthesis and growth of ZnO nanoparticles. J. Phys. Chem.

B, 102(29), 5566-5572.

Moerner, W., & Fromm, D. P. (2003). Methods of single-molecule fluorescence

spectroscopy and microscopy. Rev. Sci. Instrum., 74(8), 3597-3619.

Molpeceres, J., Aberturas, M., & Guzman, M. (2000). Biodegradable nanoparticles as a

delivery system for cyclosporine: preparation and characterization. J. Microen-

capsulation, 17(5), 599-614.

Mössmer, S., Spatz, J. P., Möller, M., Aberle, T., Schmidt, J., & Burchard, W. (2000).

Solution Behavior of Poly(styrene)-block-poly(2-vinylpyridine) Micelles

Containing Gold Nanoparticles. Macromolecules, 33(13), 4791-4798. doi: 10.10

21/ma992006i

Mulfinger, L., Solomon, S. D., Bahadory, M., Jeyarajasingam, A. V., Rutkowsky, S. A.,

& Boritz, C. (2007). Synthesis and study of silver nanoparticles. J. Chem. Educ,

84(2), 322.

Nagawa, Y., Saji, Y., Chiba, S., & Yui, T. (1971). Neuromuscular blocking actions of

nereistoxin and its derivatives and antagonism by sulfhydryl compounds. Jpn. J.

Pharmacol., 21(2), 185-197.

Nahar, M., Dutta, T., Murugesan, S., Asthana, A., Mishra, D., Rajkumar, V., Jain, N. K.

(2006). Functional polymeric nanoparticles: an efficient and promising tool for

active delivery of bioactives. Crit. Rev. in Therapeutic Drug Carrier Systems,

23(4).

Naka, K., Itoh, H., & Chujo, Y. (2003). Temperature-dependent reversible self-assembly

of gold nanoparticles into spherical aggregates by molecular recognition between

pyrenyl and dinitrophenyl units. Langmuir, 19(13), 5496-5501.

145

Namera, A., Watanabe, T., Yashiki, M., Kojima, T., & Urabe, T. (1999). Simple and

sensitive analysis of nereistoxin and its metabolites in human serum using

headspace solid-phase microextraction and gas chromatography-mass spectrometry.

J. Chromatogr. Sci., 37(3), 77-82.

Nasir, A., Kausar, A., & Younus, A. (2015). A review on preparation, properties and

applications of polymeric nanoparticle-based materials. Polym. Plast. Technol.

Eng., 54(4), 325-341.

Nicholson, R. S. (1965). Theory and Application of Cyclic Voltammetry for

Measurement of Electrode Reaction Kinetics. Anal. Chem., 37(11), 1351-1355.

O‘Farrell, N., Houlton, A., & Horrocks, B. R. (2006). Silicon nanoparticles: applications

in cell biology and medicine. Int. J. Nanomed., 1(4), 451.

Odegard, G. M., Clancy, T. C., & Gates, T. S. (2005). Modeling of the mechanical

properties of nanoparticle/polymer composites. Polymer, 46(2), 553-562. doi:

http://dx.doi.org/10.1016/j.polymer.2004.11.022

Paeng, K., Richert, R., & Ediger, M. (2012). Molecular mobility in supported thin films

of polystyrene, poly (methyl methacrylate), and poly (2-vinyl pyridine) probed by

dye reorientation. Soft Matter, 8(3), 819-826.

Pal, S. L., Jana, U., Manna, P. K., Mohanta, G. P., & Manavalan, R. (2011).

Nanoparticle: An overview of preparation and characterization (2000-2010).

Park, Y., Choe, S., Lee, H., Jo, J., Park, Y., Kim, E., Jung, J. H. (2015). Advanced

analytical method of nereistoxin using mixed-mode cationic exchange solid-phase

extraction and GC/MS. Forensic Sci. Int., 252, 143-149.

Pasch, H. (2013). Hyphenated separation techniques for complex polymers. Polymer

Chemistry, 4(9), 2628-2650.

http://dx.doi.org/10.1016/j.polymer.2004.11.022

146

Patil, V., Mayya, K., Pradhan, S., & Sastry, M. (1997). Evidence for novel interdigitated

bilayer formation of fatty acids during three-dimensional self-assembly on silver

colloidal particles. J. Am. Chem. Soc., 119(39), 9281-9282.

Patrianakos, C., & Hoffmann, D. (1979). Chemical Studies on Tobacco Smoke LXIV. On

the Analysis of Aromatic Amines in Cigarette Smoke. J. Anal. Toxicol., 3(4), 150-

154. doi: 10.1093/jat/3.4.150

Pecora, R. (1979). Dynamic light scattering from polymers. Die Makromol. Chem.,

2(S19791), 73-80. doi: 10.1002/macp.1979.020021979105

Perrault, S. D., & Chan, W. C. W. (2009). Synthesis and Surface Modification of Highly

Monodispersed, Spherical Gold Nanoparticles of 50−200 nm. J. Am. Chem. Soc.,

131(47), 17042-17043. doi: 10.1021/ja907069u

Podsiadlo, P., Paternel, S., Rouillard, J.-M., Zhang, Z., Lee, J., Lee, J.-W., Kotov, N. A.

(2005). Layer-by-layer assembly of nacre-like nanostructured composites with

antimicrobial properties. Langmuir, 21(25), 11915-11921.

Podsiadlo, P., Sinani, V. A., Bahng, J. H., Kam, N. W. S., Lee, J., & Kotov, N. A. (2008).

Gold nanoparticles enhance the anti-leukemia action of a 6-mercaptopurine

chemotherapeutic agent. Langmuir, 24(2), 568-574.

Pooja, D., Panyaram, S., Kulhari, H., Rachamalla, S. S., & Sistla, R. (2014). Xanthan

gum stabilized gold nanoparticles: characterization, biocompatibility, stability and

cytotoxicity. Carbohydr. Polym., 110, 1-9.

Pooja, D., Panyaram, S., Kulhari, H., Reddy, B., Rachamalla, S. S., & Sistla, R. (2015).

Natural polysaccharide functionalized gold nanoparticles as biocompatible drug

delivery carrier. Int. J. Biol. Macromol., 80, 48-56.

Puhakainen, E. V. J., Barlow, R. D., & Salonen, J. T. (1987). An automated colorimetric

assay for urine nicotine metabolites: a suitable alternative to cotinine assays for

the assessment of smoking status. Clin. Chim. Acta, 170(2), 255-262. doi:

https://doi.org/10.1016/0009-8981(87)90135-5

147

Quake, S. R., & Scherer, A. (2000). From Micro- to Nanofabrication with Soft Materials.

Science, 290(5496), 1536-1540. doi: 10.1126/science.290.5496.1536

Rahim, S., Ali, S. A., Ahmed, F., Imran, M., Shah, M. R., & Malik, M. I. (2017).

Evaluation of morphology, aggregation pattern and size-dependent drug-loading

efficiency of gold nanoparticles stabilised with poly (2-vinyl pyridine). J.

Nanopart. Res., 19(7), 259.

Rahim, S., Khalid, S., Bhanger, M. I., Shah, M. R., & Malik, M. I. (2018). Polystyrene-

block-poly(2-vinylpyridine)-conjugated silver nanoparticles as colorimetric

sensor for quantitative determination of Cartap in aqueous media and blood

plasma. Sens. Actuators, B, 259, 878-887. doi: https://doi.org/10.1016/j.snb.2017

.12.138

Rai, M., Yadav, A., & Gade, A. (2009). Silver nanoparticles as a new generation of

antimicrobials. Biotechnol. Adv., 27(1), 76-83.

Raymond-Delpech, V., Matsuda, K., Sattelle, B. M., Rauh, J. J., & Sattelle, D. B. (2005).

Ion channels: molecular targets of neuroactive insecticides. Invertebrate Neurosci.,

5(3-4), 119-133.

Ribbe, A. E., Okumura, A., Matsushige, K., & Hashimoto, T. (2001). Element

Spectroscopic Imaging of Poly(2-vinylpyridine)-block-polyisoprene Microdomains

Containing Palladium Nanoparticles. Macromolecules, 34(23), 8239-8245. doi:

10.1021/ma0100091

Riess, G. (2003). Micellization of block copolymers. Prog. Polym. Sci., 28(7), 1107-

1170. doi: http://dx.doi.org/10.1016/S0079-6700(03)00015-7

Riess, G., & Labbe, C. (2004). Block Copolymers in Emulsion and Dispersion

Polymerization. Macromol. Rapid Commun., 25(2), 401-435. doi: 10.1002/marc.

200300048

http://dx.doi.org/10.1016/S0079-6700(03)00015-7

148

Robinson, I., Ung, D., Tan, B., Long, J., Cooper, A. I., Fernig, D. G., & Thanh, N. T. K.

(2008). Size and shape control for water-soluble magnetic cobalt nanoparticles

using polymer ligands. J. Mater. Chem., 18(21), 2453-2458.

Rogach, A. L., Eychmüller, A., Hickey, S. G., & Kershaw, S. V. (2007). Infrared‐

emitting colloidal nanocrystals: synthesis, assembly, spectroscopy, and

applications. Small, 3(4), 536-557.

Rosen, J. E., Chan, L., Shieh, D.-B., & Gu, F. X. (2012). Iron oxide nanoparticles for

targeted cancer imaging and diagnostics. Nanomed. Nanotechnol. Biol. Med.,

8(3), 275-290.

Roy, S., & Sharma, A. (2015). Self-organized morphological evolution and dewetting in

solvent vapor annealing of spin coated polymer blend nanostructures. J. Colloid

Interface Sci., 449, 215-225.

Ruparelia, J. P., Chatterjee, A. K., Duttagupta, S. P., & Mukherji, S. (2008). Strain

specificity in antimicrobial activity of silver and copper nanoparticles. Acta

Biomater., 4(3), 707-716.

Saha, K., Agasti, S. S., Kim, C., Li, X., & Rotello, V. M. (2012). Gold Nanoparticles in

Chemical and Biological Sensing. Chem. Rev., 112(5), 2739-2779. doi: 10.1021/

cr2001178

Sana Rahim, S. A. A., Farid Ahmed, Muhammad Imran, Muhammad Raza Shah,

Muhammad Imran Malik. (2017). Evaluation of morphology, aggregation pattern

and size-dependent drug-loading efficiency of gold nanoparticles stabilised with

poly (2-vinyl pyridine). J. Nanopart. Res.(19), 259. doi: doi.org/10.1007/s11051-

017-3933-4

Sarkar, B., & Alexandridis, P. (2015). Block copolymer–nanoparticle composites:

Structure, functional properties, and processing. Prog. Polym. Sci., 40, 33-62.

149

Sartor, M. Dynamic Light Scattering University of California, San Diego.

Schaaff, T. G., & Whetten, R. L. (2000). Giant gold− glutathione cluster compounds:

intense optical activity in metal-based transitions. J. Phys. Chem. B, 104(12),

2630-2641.

Schmitt, J., Decher, G., Dressick, W. J., Brandow, S. L., Geer, R. E., Shashidhar, R., &

Calvert, J. M. (1997). Metal nanoparticle/polymer superlattice films: Fabrication

and control of layer structure. Adv. Mater., 9(1), 61-65. doi: 10.1002/

adma.19970090114

Selmar, D., Radwan, A., & Nowak, M. (2015). Horizontal natural product transfer: a so

far unconsidered source of contamination of plant-derived commodities. J.

Environ. Anal. Toxicol., 5(287), 2161-0525.1000287.

Shan, J., & Tenhu, H. (2007). Recent advances in polymer protected gold nanoparticles:

synthesis, properties and applications. Chem. Commun.(44), 4580-4598. doi:

10.1039/B707740H

Shenhar, R., Norsten, T. B., & Rotello, V. M. (2005). Polymer‐Mediated Nanoparticle

Assembly: Structural Control and Applications. Adv. Mater., 17(6), 657-669.

Shenton, W., Davis, S. A., & Mann, S. (1999). Directed Self‐Assembly of Nanoparticles

into Macroscopic Materials Using Antibody–Antigen Recognition. Adv. Mater.,

11(6), 449-452.

Shimmin, R. G., Schoch, A. B., & Braun, P. V. (2004). Polymer Size and Concentration

Effects on the Size of Gold Nanoparticles Capped by Polymeric Thiols.

Langmuir, 20(13), 5613-5620. doi: 10.1021/la036365p

Shiraishi, Y., Ikeda, M., Tsukamoto, D., Tanaka, S., & Hirai, T. (2011). One-pot

synthesis of imines from alcohols and amines with TiO 2 loading Pt nanoparticles

under UV irradiation. Chem. Commun., 47(16), 4811-4813.

150

Shukla, R., Bansal, V., Chaudhary, M., Basu, A., Bhonde, R. R., & Sastry, M. (2005).

Biocompatibility of gold nanoparticles and their endocytotic fate inside the

cellular compartment: a microscopic overview. Langmuir, 21(23), 10644-10654.

Sims, M. J., Rees, N. V., Dickinson, E. J. F., & Compton, R. G. (2010). Effects of thin-

layer diffusion in the electrochemical detection of nicotine on basal plane pyrolytic

graphite (BPPG) electrodes modified with layers of multi-walled carbon nanotubes

(MWCNT-BPPG). Sens. Actuators, B, 144(1), 153-158. doi: https://doi.org

/10.1016/j.snb.2009. 10.055

Sohn, B. H., & Seo, B. H. (2001). Fabrication of the multilayered nanostructure of

alternating polymers and gold nanoparticles with thin films of self-assembling

diblock copolymers. Chem. Mater., 13(5), 1752-1757.

Solomon, S. D., Bahadory, M., Jeyarajasingam, A. V., Rutkowsky, S. A., Boritz, C., &

Mulfinger, L. (2007). Synthesis and Study of Silver Nanoparticles. J. Chem.

Educ., 84(2), 322. doi: 10.1021/ed084p322

Sparks, T. C., & Nauen, R. (2015). IRAC: Mode of action classification and insecticide

resistance management. Pestic. Biochem. Physiol., 121, 122-128. doi: http://dx.doi.

org/10.1016/j.pestbp.2014.11.014

Spatz, J. P., Mößmer, S., & Möller, M. (1996). Mineralization of Gold Nanoparticles in a

Block Copolymer Microemulsion. Chem. Eur. J., 2(12), 1552-1555. doi: 10.1002/

chem.19960021213

Speight, J. G. (2010). Handbook of industrial hydrocarbon processes: Gulf Professional

Publishing.

Suffredini, H. B., Santos, M. C., De Souza, D., Codognoto, L., Homem‐de‐Mello, P.,

Honório, K. M., Avaca, L. A. (2005). Electrochemical behavior of nicotine

studied by voltammetric techniques at boron‐doped diamond electrodes. Anal.

Lett., 38(10), 1587-1599.

151

Sun, S., & Zeng, H. (2002). Size-controlled synthesis of magnetite nanoparticles. JACS,

124(28), 8204-8205.

Švorc, Ľ., Stanković, D. M., & Kalcher, K. (2014). Boron-doped diamond

electrochemical sensor for sensitive determination of nicotine in tobacco products

and anti-smoking pharmaceuticals. Diamond Relat. Mater., 42(Supplement C), 1-

7. doi: https://doi.org/10.1016/j.diamond.2013.11.012

Tan, E. P. S., & Lim, C. T. (2004). Physical properties of a single polymeric nanofiber.

Appl. Phys. Lett., 84(9), 1603-1605. doi: doi:http://dx.doi.org/10.1063/1.1651643

Thesleff, S. (1955). The Mode of Neuromuscular Block Caused by Acetylcholine,

Nicotine, Decamethonium and Suecinylcholine1. Acta Physiol. Scand., 34(2-3),

218-231. doi: 10.1111/j.1748-1716.1955.tb01242.x

Thurmond, K. B., Kowalewski, T., & Wooley, K. L. (1997). Shell Cross-Linked Knedels:

A Synthetic Study of the Factors Affecting the Dimensions and Properties of

Amphiphilic Core-Shell Nanospheres. J. Am. Chem. Soc., 119(28), 6656-6665.

doi: 10.1021/ja9710520

Torrisi, V., Ruffino, F., Licciardello, A., Grimaldi, M. G., & Marletta, G. (2011).

Memory effects in annealed hybrid gold nanoparticles/block copolymer bilayers.

Nanoscale Res. Lett., 6(1), 167. doi: 10.1186/1556-276x-6-167

Toshima, N., & Yonezawa, T. (1998). Bimetallic nanoparticles—novel materials for

chemical and physical applications. New J. Chem., 22(11), 1179-1201.

Trathnigg, B. (1995). Determination of MWD and chemical composition of polymers by

chromatographic techniques. Prog. Polym. Sci., 20(4), 615-650. doi:

http://dx.doi.org/10.1016/0079-6700(95)00005-Z

Turkevich, J., Stevenson, P. C., & Hillier, J. (1951). A study of the nucleation and growth

processes in the synthesis of colloidal gold. Discuss. Faraday Soc., 11, 55-75.

http://dx.doi.org/10.1063/1.1651643

http://dx.doi.org/10.1016/0079-6700(95)00005-Z

152

Tyagi, H., Kushwaha, A., Kumar, A., & Aslam, M. (2011a). pH-dependent synthesis of

stabilized gold nanoparticles using ascorbic acid. Int. J. Nanosci., 10(04n05), 857-

860. doi: 10.1142/s0219581x11009301

Umali, A. P., & Anslyn, E. V. (2010). A general approach to differential sensing using

synthetic molecular receptors. Curr. Opin. Chem. Biol., 14(6), 685-692.

Ung, D., Tung, L. D., Caruntu, G., Delaportas, D., Alexandrou, I., Prior, I. A., & Thanh,

N. T. (2009). Variant shape growth of nanoparticles of metallic Fe–Pt, Fe–Pd and

Fe–Pt–Pd alloys. CrystEngComm, 11(7), 1309-1316.

Vaia, R. A., & Maguire, J. F. (2007). Polymer Nanocomposites with Prescribed

Morphology: Going beyond Nanoparticle-Filled Polymers. Chem. Mater., 19(11),

2736-2751. doi: 10.1021/cm062693+

Verploegen, E., Mondal, R., Bettinger, C. J., Sok, S., Toney, M. F., & Bao, Z. (2010).

Effects of thermal annealing upon the morphology of polymer–fullerene blends.

Adv. Funct. Mater., 20(20), 3519-3529.

Vivek, C., Veeraiah, K., Padmavathi, P., Rao, H. D., & Bramhachari, P. V. (2016). Acute

toxicity and residue analysis of cartap hydrochloride pesticide: Toxicological

implications on the fingerlings of fresh water fish Labeo rohita. Biocatal. Agric.

Biotechnol.,7(SupplementC),193-201.doi:https://doi.org/10.1016/j.bcab.

2016.06.005

Voulgaris, D., Tsitsilianis, C., Grayer, V., Esselink, F. J., & Hadziioannou, G. (1999).

Amphiphile micelles formed by polystyrene/poly(2-vinyl pyridine) heteroarm star

copolymers in toluene. Polymer, 40(21), 5879-5889. doi: http://dx.doi.org/10.

1016/S0032-3861(98)00815-5

Walker, C. H., St. John, J. V., & Wisian-Neilson, P. (2001). Synthesis and Size Control

of Gold Nanoparticles Stabilized by Poly(methylphenylphosphazene). J. Am.

Chem. Soc., 123(16), 3846-3847. doi: 10.1021/ja005812+

153

Wang, H., Winnik, M. A., & Manners, I. (2007). Synthesis and Self-Assembly of

Poly(ferrocenyldimethylsilane-b-2-vinylpyridine) Diblock Copolymers. Macro-

molecules, 40(10), 3784-3789. doi: 10.1021/ma062728r

Weibel, M., Caseri, W., Suter, U. W., Kiess, H., & Wehrli, E. (1991). Preparation of

polymer nanocomposites with ―ultrahigh‖ refractive index. Polym. Adv. Technol.,

2(2), 75-80.

Weiss, S. (1999). Fluorescence spectroscopy of single biomolecules. Science, 283(5408),

1676-1683.

Wu, C., Siems, W. F., Hill, J. H. H., & Hannan, R. M. (1998). Analytical determination

of nicotine in tobacco by supercritical fluid chromatography–ion mobility

detection. J. Chromatogr. A, 811(1), 157-161. doi: https://doi.org/10.1016/S0021-

9673(98)00223-4

Wu, N., Zheng, A., Huang, Y., & Liu, H. (2007). Morphology of poly (styrene‐block‐

dimethylsiloxane) copolymer films. J. Appl. Polym. Sci., 104(2), 1010-1018.

Xia, Y., Kim, E., Zhao, X.-M., Rogers, J. A., Prentiss, M., & Whitesides, G. M. (1996).

Complex Optical Surfaces Formed by Replica Molding Against Elastomeric

Masters. Science, 273(5273), 347-349. doi: 10.1126/science.273.5273.347

Xue, K., Zhou, S., Shi, H., Feng, X., Xin, H., & Song, W. (2014). A novel amperometric

glucose biosensor based on ternary gold nanoparticles/polypyrrole/reduced

graphene oxide nanocomposite. Sens. Actuators B, 203, 412-416.

Yildiz, D. (2004). Nicotine, its metabolism and an overview of its biological effects.

Toxicon, 43(6), 619-632.

Youk, J. H., Park, M.-K., Locklin, J., Advincula, R., Yang, J., & Mays, J. (2002).

Preparation of Aggregation Stable Gold Nanoparticles Using Star-Block

Copolymers. Langmuir, 18(7), 2455-2458. doi: 10.1021/la015730e

154

Yu, Y. Y., Chien, W. C., & Chen, S. T. (2008). Preparation and morphology of

amphiphilic polystyrene–poly (2‐vinylpyridine) heteroarm star copolymers

prepared by ATRP. Polym. Int., 57(12), 1369-1376.

Yuan, P., Ma, R., & Xu, Q. (2016). Highly sensitive and selective two-photon sensing of

cartap using Au@Ag core-shell nanoparticles. Sci. China Chem., 59(1), 78-82.

doi: 10.1007/s11426-015-5532-5

Yusa, S.-i., Fukuda, K., Yamamoto, T., Iwasaki, Y., Watanabe, A., Akiyoshi, K., &

Morishima, Y. (2007). Salt Effect on the Heat-Induced Association Behavior of

Gold Nanoparticles Coated with Poly(N-isopropylacrylamide) Prepared via

Reversible Addition−Fragmentation Chain Transfer (RAFT) Radical

Polymerization. Langmuir, 23(26), 12842-12848. doi: 10.1021/la702741q

Zhang, H., Liu, Y., Yao, D., & Yang, B. (2012). Hybridization of inorganic nanoparticles

and polymers to create regular and reversible self-assembly architectures. Chem.

Soc. Rev., 41(18), 6066-6088.

155

LIST OF PUBLICATIONS

Sana Rahim, Sadia Khalid, Muhammad Iqbal Bhanger, Muhammad Raza Shah,

Muhammad ImranMalik, ―Polystyrene-block-poly(2-vinylpyridine)-conjugated

silver nanoparticles as colorimetric sensor for quantitative determination of

Cartap in aqueous media and blood plasma‖, Sensors and Actuators B: Chemical,

2018

Sana Rahim, Syed Abid Ali, Farid Ahmed, Muhammad Imran, Muhammad Raza

Shah, Muhammad Imran Malik, ―Evaluation of morphology, aggregation pattern

and size-dependent drug-loading efficiency of gold nanoparticles stabilised with

poly (2-vinyl pyridine)‖, Journal of Nanoparticle Research, 2017

Muhammad Khurram Tufail, Rubina Abdul-Karim, Sana Rahim, Syed Ghulam

Musharraf and Muhammad Imran Malik, ―Analysis of individual block length of

Amphiphilic di- & tri-block copolymers containing poly(ethylene oxide) and

poly(methyl methacrylate)‖, RSC Adv., 2017

Muhammad Imran Malik, Muhammad Irfan, Akbar Khan, Sana Rahim, Rubina

Abdul-Karim, Jamshed Hashim, ―Alkylene oxide poylmerizations: identification of

side reactions and by-products‖, Journal of Polymer Research, 2016.

Sana Rahim, Asma Rauf, Saba Rauf, Muhammad Iqbal Bhanger, Muhammad Raza

Shah, Muhammad Imran Malik, ―Enhanced Electrochemical Response of Modified

Glassy Carbon Electrode by poly(2-vinlypyridine-b-methyl methacrylate)

Conjugated Gold Nanoparticles for Detection of Nicotine‖, (Submitted)

Sana Rahim, Muhammad Raza Shah, Muhammad Imran Malik, ―Selectivity of

thin films of poly(2-vinylpyridine-block-methyl methacrylate) copolymers: an

AFM study‖, (In progress)

https://www.sciencedirect.com/science/article/pii/S0925400517324711#!





https://www.sciencedirect.com/science/journal/09254005

https://link.springer.com/journal/11051

Documents

Submitted by Sana Rahim